Lipase variants for pharmaceutical use

ABSTRACT

The pharmaceutical use of lipases related to the  Thermomyces lanuginosus  ( Humicola lanuginosa ) lipase comprising amino acids 1-269 of SEQ ID NO: 2, optionally in combination with a protease and/or an amylase. Examples of medical indications are: Treatment of digestive disorders, pancreatic exocrine insufficiency (PEI), pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II. The lipases of the invention have, e.g., an improved digestion performance in vitro, an improved activity at a pH in the neutral range, an improved stability at low pH, an are stable against protease-degradation, and/or are stable in the presence of pepsin and bile salts. The invention also relates to methods of determining digestion performance in vitro of lipases, as well as to certain novel variants of the lipase of  T. lanuginosus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2007/087168 filed 12 Dec. 2007, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. provisional application No.60/871,196 filed 21 Dec. 2006, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a lipase for use as a medicament, whichlipase (a) has at least 50% identity to the sequence of amino acids 1 to269 of SEQ ID NO: 2; (b) has lipase activity; and which (c) as comparedto the sequence of amino acids 1-269 of SEQ ID NO:2, comprisessubstitutions N33Q, T231R, and N233R, as well as at least one additionalsubstitution selected from the following: E1*,D,N; Q4H,P,R; D5E; N8L,Q;Q9H; F10L; N11C,D,H,L,P,Q,R,S; G23E; N26A,H,I; D27I,N,Q,R,S,V; P29T;A30T,V; T37K,M; G38A,D,F,H,I,K,L,M,N,P,Q,S,T,W,Y; N39H,S; E43K; K46M;A49T; L52I,R; E56K,Q,R,S; D57G,N; V60E,S; G61R; V63R; A68V; L69I;N71I,S; N73Q,Y; I76T; R84E; I86F,L; E87A,H,K,R; 190L,V;G91A,C,E,F,K,L,M,N,S,T,V,W,Y; L93*,F; N94*,K,Q,R,S; F95*;D96*,E,G,N,R,S,W,Y; L97M,Q; K98I,T; E99D; N101Q; D102E,G,Y; R108M;G109A; D111A,E,N,S; G112A; T114I; S115L;W117C,D,E,F,G,H,I,K,L,P,S,T,V,Y; D122E,N; Q126L; V128A; D130H; H135D;P136H; Y138F; V141E,L; A150V; V154F,I,L; A155V; G156R; G161A,E;N162G,S,T; G163A,C,D,E,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; D167E; V168M;V176A,D,F,G,H,I,K,M,N,Q,T,W; G177A; R179T; L185M; G190C,D; N200Q,S;R205I; L206F; E210D,R,V,Y; S216P; E219D; G225P; T226N; L227F,G; P229R;E239D; G240L; D242E; T244S; G246A; Q249R; N251Q,S;D254A,G,I,K,L,M,N,R,Q,S,Y; I255A,F; P256A,F,G,H,I,L,M,N,Q,S,T,V,W,Y; andL269F,H.

The invention also relates to pharmaceutical compositions comprisingthese lipases, as well as to some of these lipases as such.

The invention furthermore relates to methods of determining, andoptionally comparing, digestion performance in vitro of lipases.

The lipases of the invention may be used in combination with a proteaseand/or an amylase. Examples of medical indications are: Treatment ofdigestive disorders, pancreatic exocrine insufficiency (PEI),pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type II.

The lipase of SEQ ID NO: 2 is a wild type lipase derived from Humicolalanuginosa DSM 4109 (synonym: Thermomyces lanuginosus).

BACKGROUND ART

U.S. Pat. No. 5,614,189 (EP 600868 B1) describes the use of, i.a., alipase derived from Humicola lanuginosa in pancreatic enzyme replacementtherapy, for example in the treatment of patients suffering from cysticfibrosis. This lipase is from Humicola lanuginosa DSM 4109 and has theamino acid sequence of amino acids 1-269 of SEQ ID NO: 2.

WO 92/05249, WO 92/19726, WO 94/25577, WO 95/09909, WO 95/22615, WO97/04079, WO 97/07202, WO 99/42566, WO 00/32758, WO 00/60063, WO01/83559, WO 01/83559, WO 2002/055679, WO 2002/062973, WO 2002/062973,WO 2004/099400, and WO 2004/111216 describe a number of variants of SEQID NO: 2, but not the pharmaceutical use thereof.

WO 2006/136159 describes the pharmaceutical use of the lipase havingamino acids 1-269 of SEQ ID NO: 1, as well as variant N33Q thereof.

There is a need in the art for improved lipases for pharmaceutical use.

SUMMARY OF THE INVENTION

The present invention provides improved lipases for pharmaceutical use.Preferably, the enzymes for use according to the invention have animproved efficacy in vivo and/or in vitro; an improved activity; animproved stability; are stable against degradation by proteases; arestable in the presence of bile salts; and/or have a reducedallergenicity. More preferably, the lipases of the invention have animproved digestion performance in vitro, as compared to a referencelipase having the sequence of SEQ ID NO: 2 with the followingsubstitutions: N33Q+T231R+N233R.

The present invention relates to a lipase for use as a medicament, whichlipase (a) has at least 50% identity to the sequence of amino acids 1 to269 of SEQ ID NO: 2; (b) has lipase activity; and which (c) as comparedto the sequence of amino acids 1-269 of SEQ ID NO:2, comprisessubstitutions N33Q, T231R, and N233R, as well as at least one additionalsubstitution selected from the following: E1*,D,N; Q4H,P,R; D5E; N8L,Q;Q9H; F10L; N11C,D,H,L,P,Q,R,S; G23E; N26A,H,I; D27I,N,Q,R,S,V; P29T;A30T,V; T37K,M; G38A,D,F,H,I,K,L,M,N,P,Q,S,T,W,Y; N39H,S; E43K; K46M;A49T; L52I,R; E56K,Q,R,S; D57G,N; V60E,S; G61R; V63R; A68V; L69I;N71I,S; N73Q,Y; I76T; R84E; I86F,L; E87A,H,K,R; 190L,V;G91A,C,E,F,K,L,M,N,S,T,V,W,Y; L93*,F; N94*,K,Q,R,S; F95*;D96*,E,G,N,R,S,W,Y; L97M,Q; K98I,T; E99D; N101Q; D102E,G,Y; R108M;G109A; D111A,E,N,S; G112A; T114I; S115L;W117C,D,E,F,G,H,I,K,L,P,S,T,V,Y; D122E,N; Q126L; V128A; D130H; H135D;P136H; Y138F; V141E,L; A150V; V154F,I,L; A155V; G156R; G161A,E;N162G,S,T; G163A,C,D,E,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; D167E; V168M;V176A,D,F,G,H,I,K,M,N,Q,T,W; G177A; R179T; L185M; G190C,D; N200Q,S;R205I; L206F; E210D,R,V,Y; S216P; E219D; G225P; T226N; L227F,G; P229R;E239D; G240L; D242E; T244S; G246A; Q249R; N251Q,S;D254A,G,I,K,L,M,N,R,Q,S,Y; I255A,F; P256A,F,G,H,I,L,M,N,Q,S,T,V,W,Y; andL269F,H.

The invention furthermore relates to the use of such lipases for themanufacture of a medicament for the treatment of digestive disorders,PEI, pancreatitis, cystic fibrosis, diabetes type I, and/or diabetestype II, these uses optionally further comprising the use of a protease,and/or an amylase; as well as to such lipases for use in the treatmentof these conditions, optionally in combination with a protease and/or anamylase.

The invention furthermore relates to a pharmaceutical compositioncomprising such lipases, together with at least one pharmaceuticallyacceptable auxiliary material, optionally including a protease and/or anamylase.

The invention also relates to a method for the treatment of digestivedisorders, PEI, pancreatitis (acute and/or chronic), cystic fibrosis,diabetes type I, and/or diabetes type II, by administering atherapeutically effective amount of such lipases, optionally togetherwith a protease and/or an amylase.

Finally, the invention relates to methods for determining, andoptionally comparing, lipase digestion performances in vitro; as well asto certain lipases as such, e.g.:

A lipase which (a) has at least 50% identity to the sequence of aminoacids 1-269 of SEQ ID NO: 2; (b) has lipase activity; and which, (c) ascompared to the sequence of amino acids 1-269 of SEQ ID NO: 2, comprisesa substitution in at least one position selected from the following: 9,30, 38, 39, 63, 112, 114, 115, 117, 122, 128, 130, 136, 154, 155, 156,161, 163, 168, 185, 190, 239, and 246; and

A lipase which (a) has at least 50% identity to the sequence of aminoacids 1-269 of SEQ ID NO: 2; (b) has lipase activity; and which,

(c1) as compared to the sequence of amino acids 1-269 of SEQ ID NO: 2,comprises at least one substitution selected from the following: E1N;Q4H; N8L,Q; Q9H; N11C,D,H,L,P,S; G23E; D27I; P29T; A30T,V; T37K,M;G38A,D,F,H,I,K,L,M,N,P,Q,S,T,W,Y; N39H,S; D57N; G61R; V63R; N71I,S;N73Q,Y; I76T; I86F,L; E87H; G91F,K,L,M,V,Y; N94Q; F95*; D96*; N101Q;D111E; G112A; T114I; S115L; W117C,D,E,F,G,H,I,K,L,P,S,T,V,Y; D122E,N;Q126L; V128A, D130H, H135D, P136H; V141E,L; V154F,I,L; A155V; G156R;G161A,E; N162G,S; G163A,C,D,E,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; V168M; L185M;G190C,D; R205I; G240L; G246A; N251Q,S; and L269F,H; or which,

(c2) as compared to the sequence of amino acids 1-269 of SEQ ID NO: 2,comprises at least one of the following amino acids at the indicatedposition: 1N; 4H; 8L,Q; 9H; 11C,D,H,L,P,S; 23E; 27I; 29T; 30T,V; 37K,M;38A,D,F,H,I,K,L,M,N,P,Q,S,T,W,Y; 39H,S; 57N; 61R; 63R; 71I,S; 73Q,Y;76T; 86F,L; 87H; 91F,K,L,M,V,Y; 94Q; 95*; 96*; 101Q; 111E; 112A; 114I;115L; 117C,D,E,F,G,H,I,K,L,P,S,T,V,Y; 122E,N; 126L; 128A, 130H, 135D,136H; 141E,L; 154F,I,L; 155V; 156R; 161A,E; 162G,S;163A,C,D,E,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 168M; 185M; 190C,D; 205I; 240L;246A; 251Q,S; and 269F,H.

DETAILED DESCRIPTION OF THE INVENTION

Lipases

A lipase is a polypeptide having lipase activity. In what follows, thelipase for use in the compositions, methods and uses of the invention isreferred to as the lipase of the invention. The lipase of the inventionmay be a carboxylic ester hydrolase EC 3.1.1.-, which includesactivities such as EC 3.1.1.3 triacylglycerol lipase, EC 3.1.1.4phospholipase A2, EC 3.1.1.5 lysophospholipase, EC 3.1.1.26galactolipase, EC 3.1.1.32 phospholipase A1, EC 3.1.1.73 feruloylesterase. In a particular embodiment, the lipase is an EC 3.1.1.3triacylglycerol lipase. In another particular embodiment, the lipase hasEC 3.1.1.4 phospholipase A2 activity, i.e., catalyzes the reaction:Phosphatidylcholine+H(2)O=1-acylglycerophosphocholine+a carboxylate(removes the fatty acid attached to the 2-position). In a still furtherparticular embodiment, the lipase has EC 3.1.1.32 phospholipase A1activity, i.e., catalyzes the reaction:Phosphatidylcholine+H(2)O=2-acylglycerophosphocholine+a carboxylate.

The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, AcademicPress, San Diego, Calif., including supplements 1-5 published in Eur. J.Biochem., 1994, 223: 1-5; Eur. J. Biochem., 1995, 232: 1-6; Eur. J.Biochem., 1996, 237: 1-5; Eur. J. Biochem., 1997, 250: 1-6; and Eur. J.Biochem., 1999, 264: 610-650; respectively. The nomenclature isregularly supplemented and updated; see, e.g., the World Wide Web atwww.chem.qmw.ac.uk/iubmb/enzyme/index.html.

The lipase of the invention may be a variant of a parent lipase.

Variant

The term variant is defined herein as a lipase comprising one or morealterations, such as substitutions, insertions, deletions, and/ortruncations of one or more specific amino acid residues at one or morespecific positions in the polypeptide, as compared to a parent lipase.

Parent Lipase

The term parent lipase refers to the polypeptide with which a variant iscompared and aligned. A particular example of a parent lipase is thelipase to which modifications, e.g., substitution(s), insertion(s),deletion(s), and/or truncation(s), are made to produce the lipasevariants of the present invention. The parent may be a naturallyoccurring (wild-type) lipase, or it may be a variant thereof, preparedby any suitable means. A parent may also be an allelic variant which isa polypeptide encoded by any of two or more alternative forms of a geneoccupying the same chromosomal locus.

In a particular embodiment, the parent lipase is a fungal lipase with anamino acid sequence having at least 50% identity to the sequence ofamino acids 1-269 of the T. lanuginosus lipase shown in SEQ ID NO: 2.The parent lipase may be a yeast lipase such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or, more preferably, a filamentous fungal lipase such as anAcremonium, Aspergillus, Aureobasidium, Cryptococcus, Filobasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermalipase—or a variant of any of these. A preferred parent lipase is anascomycete lipase, preferably derived from a strain of Humicola,Talaromyces or Thermomyces, e.g., from a strain of Humicola fuscoatra,Humicola grisea, Humicola insolens, Humicola lutea, Humicola nigrescens,Humicola sp., Humicola lanuginosa (Thermomyces lanoginosus), Thermomycesibadanensis, Thermomyces veffucosus, Talaromyces thermophilus,Talaromyces emersonii, or Talaromyces byssochlamydoides or variants ofany of these. In a particular embodiment, the parent lipase is (i) theHumicola lanuginosa lipase having amino acids 1 to 269 of SEQ ID NO: 2,or (ii) a variant thereof.

Nomenclature of Lipase Variants

In the present invention, a specific numbering of amino acid residuepositions in the lipase variants is employed. By aligning the amino acidsequences of known lipases, it is possible to designate an amino acidposition number to any amino acid residue in any lipase enzyme.

Using the numbering system originating from the amino acid sequence ofthe lipase disclosed in SEQ ID NO: 2, aligned with the amino acidsequence of another lipase using the alignment procedure hereindescribed, it is possible to indicate the position of each amino acidresidue in any other lipase. Accordingly, for any lipase of theinvention which is compared with the sequence of amino acids 1-269 ofSEQ ID NO: 2, each position and/or substitution corresponds to aposition of amino acids 1-269 of SEQ ID NO: 2.

In describing the various lipase variants of the present invention, thenomenclature described below is adapted for ease of reference. In allcases, the accepted IUPAC single letter or triple letter amino acidabbreviation is employed.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of asparagine with isoleucine at position 26 is designatedas N26I. Multiple mutations are separated by addition marks (+), e.g.,N33Q+E210D+T231R+N233R represent mutations at positions 33, 210, 231,and 233 substituting asparagine (N) with glutamine (Q), glutamic acid(E) with aspartic acid (D), threonine (T) with arginine (R), andasparagine (N) with arginine (R), respectively.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position,*. Accordingly, the deletion of glutamic acid (E)at position 1 is designated as “E1*”. Multiple deletions are separatedby addition marks (“+”), e.g. the deletion of leucine (L), asparagine(N), phenylalanine (F), and aspartic acid (D) in positions 93, 94, 95,and 96, respectively, is designated as “L93*+N94*+F95*+D96*”.

Accordingly, for the present purposes, a deletion can in fact beconsidered an example of a substitution, viz. a substitution of theoriginal amino acid with nothing. The following variant of the lipase ofamino acids 1-269 of SEQ ID NO: 2 can therefore be said to include 11substitutions in total: 7 substitutions into another amino acid, and 4substitutions into nothing, i.e., 4 deletions:D27R+N33Q+G91A+L93*+N94*+F95*+D96*+D111A+T231R+N233R+P256T.

Therefore, when a specific amino acid can be substituted with two ormore different amino acids or deleted, this is indicated as asubstitution, where the alternative substituents including the deletionare separated by commas. For example, the designation “E1*,D,N” meansthat glutamine at position 1 in the parent lipase (E1) may besubstituted with nothing (i.e., deleted) (*), substituted with asparticacid (D), or substituted with asparagine (N).

Alignment and Identity Calculation

This section applies to the lipases, amylases, and proteases of thepresent invention (the enzymes of the invention).

The relatedness between two amino acid sequences is described by theparameter “identity”.

For purposes of the present invention, the alignment of two amino acidsequences is determined by using the Needle program from the EMBOSSpackage (available on the Internet at emboss.org) version 2.8.0. TheNeedle program implements the global alignment algorithm described inNeedleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453. The substitutionmatrix used is BLOSUM62, gap opening penalty is 10, and gap extensionpenalty is 0.5.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”; e.g. variant LVA023 having the sequenceof amino acids 1-269 of SEQ ID NO: 2 with the following foursubstitutions: N33Q+E210D+T231R+N233R) and a different amino acidsequence (“foreign sequence”; e.g. amino acids 1-269 of SEQ ID NO: 2) iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence” or thelength of the “foreign sequence”, whichever is the shortest. The resultis expressed in percent identity.

An exact match occurs when the “invention sequence” and the “foreignsequence” have identical amino acid residues in the same positions ofthe overlap (in the alignment example below this is represented by “|”)The length of a sequence is the number of amino acid residues in thesequence (e.g. the length of SEQ ID NO: 2 is 269).

In the, purely hypothetical, alignment example below, the overlap is theamino acid sequence “HTWGER-NL” of Sequence 1; or the amino acidsequence “HGWGEDANL” of Sequence 2. In the example a gap is indicated bya “-”.

Hypothetical alignment example:

Accordingly, the percentage of identity of Sequence 1 to Sequence 2 is6/12=0.5, corresponding to 50%.

In a particular embodiment, the percentage of identity of an amino acidsequence of a polypeptide with, or to, amino acids 1-269 of SEQ ID NO: 2is determined by i) aligning the two amino acid sequences using theNeedle program, with the BLOSUM62 substitution matrix, a gap openingpenalty of 10, and a gap extension penalty of 0.5; ii) counting thenumber of exact matches in the alignment; iii) dividing the number ofexact matches by the length of the shortest of the two amino acidsequences, and iv) converting the result of the division of iii) intopercentage.

In a preferred embodiment, the lipase of the invention is at least 51%identical to the lipase having amino acids 1-269 of SEQ ID NO: 2. Inadditional preferred embodiments, it is at least 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, or at least 60% identical to the lipase having aminoacids 1-269 of SEQ ID NO: 2. In additional preferred embodiments, thepercentage of identity is at least 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, or at least 70%. In further preferred embodiments, thepercentage of identity is at least 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, or at least 80%. In additional preferred embodiments, thepercentage of identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, or at least 90%. In additional more preferred embodiments, thepercentage of identity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least 99%.

In another preferred embodiment, the parent lipase is at least 51%identical to the lipase having amino acids 1-269 of SEQ ID NO: 2. Inadditional preferred embodiments, it is at least 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least 99% identical to the lipase having amino acids 1-269 ofSEQ ID NO: 2.

Improved Properties

The present invention provides improved lipases for pharmaceutical use.Preferably, the enzymes for use according to the invention have animproved efficacy in vivo and/or in vitro; an improved activity; animproved stability; are stable against degradation by proteases; arestable in the presence of bile salts; and/or have a reducedallergenicity.

The lipases of the invention are preferably purified, more preferably tohomogeneity, e.g., as described in Example 5 of WO 2006/136159. Purifiedlipase preparations may be analyzed by SDS-PAGE, and the lipase may beidentified as the main protein band at 30-40 kDa. By densitometerscanning of coomassie-stained SDS-PAGE gels this band preferablyconstitutes 90-97% of the protein spectrum. The densitometer is, e.g., aGS-800 calibrated densitometer from BIO-RAD.

The lipase of the invention has an improved digestion performance ascompared to a reference lipase, preferably an improved digestionperformance in vitro.

The digestion performance is preferably determined using (I) a digestionmodel, (II) by determining stability at pH 3 in the presence of pepsin,and/or (III) by determining the activity at pH 5 in the presence of bilesalts.

For each of the methods (I), (II), and (III), which are furtherdiscussed below, lipase variant N33Q+T231R+N233R of SEQ ID NO: 2 is apreferred example of a reference lipase (disclosed in WO 2006/136159).Other examples of reference lipases are: The lipase of SEQ ID NO: 1(N33Q+T231R of SEQ ID NO: 2), and the lipase of SEQ ID NO: 2. A stillfurther example of a reference lipase is the average of the lipase ofamino acids +1 to +269 of SEQ ID NO: 1 and variant N33Q thereof.

The digestion model (I) represents a novel method of determiningdigestion performance, which comprises the following steps:

a) selecting a reference lipase;

b) mixing 100 parts per volume of a diet with 20 parts per volume ofpepsin and 30 parts per volume of the lipase or the reference lipase,respectively;

c) adding i) 0 or ii) 10 parts per volume of buffer (0.8 M MES, 0.8 Msodium acetate, 0.8 M imidazole, pH 7.0), wherein step i) may bereferred to as a gastric step of pH 3, and step ii) may be referred toas a gastric step of pH 5;

d) incubating for 1 hour at 37° C. with shaking;

e) adding 20 parts per volume of bile salts, as well as i) 25 or ii) 15parts per volume of buffer (0.8 M MES, 0.8 M sodium acetate, 0.8 Mimidazole, pH 7.0), wherein step i) corresponds to a gastric step of pH3, and step ii) corresponds to a gastric step of pH 5;

f) incubating for 2 hours at 37° C. with agitation;

g) adding 50 parts per volume of 10% Triton-X100 in 1 M phosphoric acid;

h) determining the amount of free fatty acids;

i) fitting the dose response curves to the equation:FFA=FFAmax*[E]/([E]+K)where FFA is the amount of released free fatty acids, FFAmax is themaximal amount of free fatty acids that the lipases can liberate fromthe diet, [E] is the lipase concentration, and K is the lipaseconcentration that liberates half of FFAmax; and

j) calculating an Improvement Factor (IF) as follows:IF=K(ref)/K(lipase),where K(ref) is the concentration of the reference lipase that liberateshalf of FFAmax and K(lipase) is the lipase concentration that liberateshalf of FFAmax.

The digestion performance may also be determined by the novel pepsinstability test at pH 3 referred to as (II) above, which method comprisesthe following steps:

i) selecting a reference lipase;

ii) mixing 5 parts per volume of the lipase or the reference lipase,respectively, with 5 parts per volume of

-   -   a) a diluent containing 0.01% Triton-X100 and 10 mM NaCl, or    -   b) a pepsin treatment solution containing 150 ug/mL pepsin, 4 mM        CaCl₂, 0.01% Triton-X100, and 50 mM Citrate, pH 3.0,

wherein a) is referred to as untreated sample, and b) is referred to aspepsin-treated sample;

iii) incubating the samples of step ii) for 3 hours at 20° C.;

iv) adding to each sample of step iii) 55 parts per volume of substratecontaining 1 mM PNP-Palmitate, 1.2% Triton-X100, 4 mM CaCl₂, 100 mMTRIS, pH 8.0, together with

-   -   a) 5 parts per volume of pepsin-treatment solution, or    -   b) 5 parts per volume of diluent

wherein a) refers to the untreated sample, and b) to the pepsin-treatedsample;

v) following degradation of the substrate by reading OD₄₀₅ of thesamples of iv) at intervals;

vi) collecting data from v) that falls in the linear range andcalculating lipase activity for the pepsin-treated sample and theuntreated sample, respectively, in mOD (milli OD) per hour;

vii) calculating % Residual lipase Activity (% RA) by dividing thelipase activity of the pepsin-treated sample with that of the untreatedsample as they result from step vi), and multiplying the result by 100;and, if desired,

viii) comparing % RA of the lipase with that of the reference lipase.

The digestion performance may also be determined by the novel bile saltactivity test at pH 5 referred to as (III) above, which method comprisesthe following steps:

i) selecting a reference lipase;

ii) mixing 10 parts per volume of the lipase or the reference lipase,respectively, with 23 parts per volume of a) water, or b) 20 mM Bilesalts, wherein a) is referred to as untreated sample, and b) is referredto as bile salts sample;

iii) adding, to each sample of ii), 200 parts per volume of substratecontaining 1 mM PNP Oleate in 25 mM Succinate, 2 mM CaCl₂, 1.2%Triton-X100, pH 5.0, and mixing;

iv) immediately after step iii), removing, from each sample, 60 partsper volume of the resulting mixture and transferring four times 15 partsper volume thereof into four separate compartments;

v) adding, after 1, 2, 3, and 4 hours, 60 parts per volume of 100 mMTRIS, pH 8.0 to the respective compartment of the four compartments ofiv), immediately reading OD 405, and, based on the linear range of the1, 2, 3, and 4 hours readings, calculating the activity in mOD/hour;

vi) dividing, for the lipase as well as the reference lipase, theactivity, obtained in step v), of the bile salts sample by the activityof the untreated sample, as also obtained in step v), to arrive at thebile salt stability ratios of the lipase and the reference lipase,respectively; and

vii) dividing the bile salt stability ratio of the lipase by the bilesalt stability ratio of the reference lipase, which resulting ratio maybe defined as the improvement factor of the lipase.

Methods (I), (II) and (III) have been found to, surprisingly, identifyimproved lipases, of which a high proportion may also be improved invivo.

Method (I)

The digestion model (referred to as (I) above) mimics digestion inmonogastric animals (such as, e.g., pig and human beings) suffering frompancreatic exocrine insufficiency.

Triton X-100 (C₁₄H₂₂O(C₂H₄O)_(n)) (CAS No. 9002-93-1) is a nonionicsurfactant which has a hydrophilic polyethylene oxide group (on averageit has 9.5 ethylene oxide units, i.e., n=9-10) and a hydrocarbonlipophilic or hydrophobic group. The hydrocarbon group is a4-(1,1,3,3-tetramethylbutyl)-phenyl group.

The term “parts per volume” preferably designates microliter which maybe abbreviated ul, uL, μl, or μL.

In particular embodiments of the in vitro digestion model, (a) thepepsin concentration is 700 mg/ml; (b) the lipase and/or the referencelipase are analyzed in 4 different concentrations, each preferably induplicate; and/or (c) the reactions take place in wells of a microtiterplate.

In additional particular embodiments of the in vitro digestion model,(d) the concentration of bile salts is 50 g/l; (e) the resulting pHafter addition of bile salts and buffer in step e) is in the range of5.7 to 6.0; and/or (f) Triton-X100 when added in step g) serves to stopthe reaction.

In still further particular embodiments of the in vitro digestion model,(g) the amount of free fatty acids is determined after appropriatedilution, such as 125-250 times, preferably in 1% Triton-X100, andpreferably determined using a NEFA C kit from Wako Chemicals, which isdescribed in Example 3); (h) the dose response curve in step i) refersto the curve showing the response, viz. the amount of free fatty acids,as a function of lipase dose; and/or (i) assuming that FFAmax isidentical for the lipases, i.e., for the reference lipase and thelipase(s) in question.

In additional particular embodiments of the in vitro digestion model,(j) Active Site Titration (AST, Example 6) is used to determine thelipase concentration; and/or (k) A₂₈₀ is used to determine the lipaseconcentration, preferably using the extinction coefficient 1.24 A₂₈₀/mg.

An improved lipase is defined as a lipase which has an improvementfactor above 1.00. In particular embodiments, (i) the improvement factoris the average improvement factor; (ii) the improvement factor is theaverage improvement factor minus the standard deviation; and/or (iii)the improvement factor is above 1.0, or above 1. Average and standarddeviation takes experimental variation into account and may becalculated as is known in the art, e.g. Standarddeviation=(Sum(IF−Avg(IF))/(n−1))^0.5, where IF is the ImprovementFactor, Avg(IF) is the average of the calculated Improvement Factors andn is the number of calculated Improvement Factors. The inverted V meansexp.

The diet of the in vitro digestion model preferably contains between 250and 400 g fat/kg, more preferably between 300 and 350 g fat/kg, mostpreferably between 313 and 340 g fat/kg. The content of carbohydrate andprotein is not as relevant, but preferably it reflects usual and typicaldietary requirements and recommendations, e.g. a carbohydrate content of250-500 g/kg, and a protein content of between 10 and 200 g/kg. The dietmay be selected from, e.g., Diet I (340 g fat/kg, 450 g carbohydrate/kg,20 g protein/kg), or Diet II (313 g fat/kg, 358 g carbohydrate/kg, and146 g protein/kg).

Diet I contains 247.2 parts per weight of cow's milk (1.5% fat), 29.9parts per weight of olive oil, 87 parts per weight of Calshake(commercially available from Fresenius Kabi and having an energy contentof 2077 kJ/g, a protein content of 4.3 g milk protein/100 g, and a fatcontent of 24.4 g fat/100 g), and 9.9 parts per weight of g Methocel(Food Grade, E5 Premium LV FG (E464); Dow). The ingredients are mixed,e.g. using an UltraTurrex (YellowLine DI 25 basic) for 2 minutes.Optionally, the diet is treated with 0.5 ug/ml of the SAVINASE 16.0 LEXprotease (commercially available from Novozymes A/S, Krogshoejvej 36,DK-2880 Bagsvaerd, Denmark) at pH 8.0 for 4 hours at 50° C. to reduceviscosity. The protease is subsequently inactivated by reducing pH to 3and incubating at 70° C. for 30 min, or 50° C. for 60 min. The term“parts by weight” preferably refers to gram (g).

Diet II contains, preferably consists of, 73 g/kg (wet weight) poultrymeal (Altromin), 73 g/kg pea meal, 73 g/kg casein (precipitated underacidic conditions, from Altromin), 290 g/kg wheat flour, 290 g/kg potatostarch, 125 g/kg lard, 76 g/kg vitamins, minerals and trace elements,and 375 g/kg cow's cream (33% fat).

Method (II)

The following are particular embodiments of the method of determiningdigestion performance by measuring the stability at pH 3 in the presenceof pepsin, which method is referred to as (II) above:

(a) The lipases used in step ii) are culture supernatants, preferablyprepared as follows: Single yeast colonies, such as colonies ofSaccharomyces cerevisiae JG169 (see e.g., U.S. Pat. No. 7,217,433),capable of expressing the lipase are picked into 1 part per volume (e.g.1 mL) of a suitable medium (e.g. the Seed Culture Medium of Example 8),and grown overnight at 30° C. and 250 rpm. Expression of the lipase isachieved by inoculating 0.020 parts per volume (e.g. 20 uL) of theresulting Seed Culture into 1 part per volume of a suitable medium (e.g.the Optimized Medium of Example 8) and cultivating at 30° C. and 250 rpmfor 4-6 days. The cultivation may be performed in e.g. microtiterplates, e.g., 24-well plates, or in shake flasks. The lipase samples maybe appropriately diluted, e.g. 25-fold, in diluent;

(b) The 3 hours of incubation of step iii) may be at room temperature;

(c) The reading of the OD₄₀₅ of step v) may e.g. take place 6 times; ande.g. as early as 15 minutes after substrate addition and as long as 18hours after substrate addition;

(d) In step vi), lipase activity is calculated for the pepsin-treatedsample and the untreated sample, respectively, in mOD (milli OD) perhour, and data from v) are collected that falls in the linear range;

(e) In step vii), calculating % Residual lipase Activity (% RA) bydividing the rate from step vi) of the pepsin-treated lipase by the rateof the untreated condition and multiplying the result by 100; and/or

(f) In step v), OD 540 is also read and used to correct for backgroundOD by subtracting the OD540 reading from the OD405 reading.

Method (III)

The following are particular embodiments of the method of determiningdigestion performance by measuring the activity at pH 5 in the presenceof bile salts (referred to as (III) above):

(a) The lipases used in step ii) are purified; (b) the lipases areappropriately diluted, typically from between 25-fold to 200-fold indiluent (such as 0.01% Triton-X100, 10 mM NaCl), e.g. to approximately 8micrograms/mL; (c) the concentration of the purified lipase samples isdetermined from the absorbance at 280 nm using the extinctioncoefficient 1.24 A₂₈₀/mg; (d) ODs between about 0.100 and 0.475 are inthe linear range; and/or (e) the bile salts are Sigma B-8756 made up indistilled water to 20 mM.

In a further particular embodiment (f) in step vi) a ratio of theactivity in the presence of bile salts at pH 5.0 is expressed as apercentage by calculating the average of all linear data obtained instep v) corrected for time and dilution for the “bile salts” activitydivided by the average of all linear data corrected for time anddilution for the “no bile salts” activity.

(A) In a first particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+E87K+T231R+N233R;-   N33Q+N94K+T231R+N233R;-   N33Q+D96Y+T231R+N233R;-   N33Q+K98I+T231R+N233R;-   A30V+N33Q+K98I+T231R+N233R;-   N33Q+E87K+D96E+T231R+N233R;-   N26I+N33Q+T231R+N233R;-   A30T+N33Q+T231R+N233R;-   N33Q+G91V+T231R+N233R;-   N33Q+G91A+T231R+N233R;-   N33Q+G91V+L97M+T231R+N233R;-   N33Q+K98I+T231R+N233R;-   N33Q+L69I+G91E+T231R+N233R;-   P29T+N33Q+T231R+N233R;-   N33Q+G91V+T231R+N233R;-   N33Q+K98I+T231R+N233R;-   N33Q+G91E+T231R+N233R; and-   N33Q+N94K+T231R+N233R.

In a second particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+K98I+T231R+N233R:-   A30V+N33Q+K98I+T231R+N233R;-   N33Q+G91V+T231R+N233R;-   N33Q+G91A+T231R+N233R;-   N33Q+G91V+L97M+T231R+N233R;-   N33Q+K98I+T231R+N233R;-   N33Q+L69I+G91E+T231R+N233R;-   P29T+N33Q+T231R+N233R;-   N33Q+G91V+T231R+N233R;-   N33Q+K98I+T231R+N233R;-   N33Q+G91E+T231R+N233R; and-   N33Q+N94K+T231R+N233R.

In a third particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+K98I+T231R+N233R;-   N33Q+G91V+T231R+N233R;-   N33Q+G91A+T231R+N233R;-   N33Q+G91V+L97M+T231R+N233R;-   N33Q+K98I+T231R+N233R;-   N33Q+L69I+G91E+T231R+N233R;-   N33Q+G91E+T231R+N233R; and-   N33Q+N94K+T231R+N233R.

In a fourth particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+G91V+T231R+N233R;-   N33Q+G91A+T231R+N233R;-   N33Q+K98I+T231R+N233R; and-   N33Q+G91E+T231R+N233R.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the four particular embodiments) allhave an improved in vitro digestion performance, i.e., an improvementfactor (IF) of at least 1.50 (or 1.5), 2.00 (or 2.0), 2.50 (or 2.5),3.00 (or 3.0), 3.50 (or 3.5), or at least 4.00 (or 4.0), preferably ofat least 5.00 (or 5.0), 6.00 (or 6.0), 7.00 (or 7.0), 8.00 (or 8.0),9.00 (or 9.0), 10.00 (or 10.0), or at least 11.00 (or 11.0). A gastricstep of pH 3 is preferably used. A preferred diet is diet II. ActiveSite Titration (AST, Example 6) and/or A₂₈₀ may be used to determine thelipase concentration, preferably using the extinction coefficient 1.24A₂₈₀/mg.

(B) In another first particular embodiment, the lipase of the inventionis selected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27V+N33Q+V60S+D96W+T231R+N233R+Q249R;-   D27V+N33Q+V60S+T231R+N233R+Q249R;-   Q9H+N33Q+D102E+T231R+N233R;-   N33Q+D111E+T231R+N233R;-   N33Q+D122E+T231R+N233R;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+P256T;-   N33Q+T231R+N233R+P256T;-   D27R+N33Q+G91A+L93*+N94*+F95*+D96*+D111A+T231R+N233R+P256T;-   N11R+N33Q+T231R+N233R;-   N33Q+N39H+T231R+N233R;-   N33Q+P229R+T231R+N233R;-   D27R+N33Q+G91N+N94R+D111A+G163K+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+G163K+T231R+N233R;-   D27R+N33Q+G91A+D96E+L97Q+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91A+D96E+L97Q+D111A+S216P+T231R+N233R+P256T;-   D27R+N33Q+G91A+D96E+D111A+T231R+N233R+D254G+P256T;-   D27R+N33Q+G91A+N94S+D111A+T231R+N233R+P256T;-   N33Q+N200S+T231R+N233R;-   N33Q+N39S+T231R+N233R;-   N33Q+E210R+T231R+N233R;-   N33Q+N39H+T231R+N233R+D254R;-   N33Q+T231R+N233R+D254R;-   N33Q+N94R+T231R+N233R;-   N33Q+D96R+T231R+N233R;-   D27N+N33Q+T231R+N233R;-   D27N+N33Q+E56R+T231R+N233R;-   N33Q+L227F+T231R+N233R;-   N33Q+N73Y+G225P+T231R+N233R;-   N33Q+G225P+T231R+N233R;-   N33Q+T231R+N233R+D254S;-   N33Q+D96G+T231R+N233R;-   N33Q+D96N+T231R+N233R+D254S;-   N33Q+T231R+N233R+D254G;-   N33Q+D130H+T231R+N233R;-   N33Q+E87A+T231R+N233R;-   N33Q+T231R+N233R+E239D;-   N33Q+D111A+T231R+N233R+D254G;-   N33Q+E210V+T231R+N233R+D254S;-   N11R+N33Q+E210V+T231R+N233R+D254S;-   N33Q+G91T+G163K+T231R+N233R+D254G;-   N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   Q4R+D27R+N33Q+G91T+N94S+D111A+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R;-   Q4R+D27R+N33Q+G91T+N94S+D111A+E210D+S216P+L227G+T231R+N233R+P256T;-   Q4R+D27Q+N33Q+G91T+N94S+D111A+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+N94S+D111A+T231R+N233R+P256T;-   N33Q+G177A+T231R+N233R;-   N33Q+T231R+N233R+G246A;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S;-   D27Q+N33Q+G91T+G163K+E219D+T231R+N233R; and-   N33Q+G91T+E219D+T231R+N233R.

In another second particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27V+N33Q+V60S+D96W+T231R+N233R+Q249R;-   D27R+N33Q+G91A+D96E+L97Q+D111A+S216P+T231R+N233R+P256T;-   D27R+N33Q+G91A+D96E+D111A+T231R+N233R+D254G+P256T;-   N33Q+N39S+T231R+N233R;-   N33Q+N94R+T231R+N233R;-   N33Q+T231R+N233R+D254S;-   N33Q+D96N+T231R+N233R+D254S;-   N33Q+E210V+T231R+N233R+D254S;-   N11R+N33Q+E210V+T231R+N233R+D254S;-   N33Q+G91T+G163K+T231R+N233R+D254G;-   N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R;-   N33Q+G91T+N94S+D111A+T231R+N233R+P256T; and-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S.

In another third particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+D96N+T231R+N233R+D254S;-   N33Q+E210V+T231R+N233R+D254S;-   N11R+N33Q+E210V+T231R+N233R+D254S;-   N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S; and-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R.

In another fourth particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the four other particularembodiments) all have an improved in vitro digestion performance, i.e.,an improvement factor (IF), preferably average IF minus standarddeviation, of above 1.00, or of at least 1.50 (or 1.5), 2.00 (or 2.0),2.50 (or 2.5), 3.00 (or 3.0), 3.50 (or 3.5), or at least 4.00 (or 4.0),preferably of at least 5.00 (or 5.0), 6.00 (or 6.0), 7.00 (or 7.0), 8.00(or 8.0), 9.00 (or 9.0), 10.00 (or 10.0), or at least 11.00 (or 11.0). Agastric step of pH 3 is preferably used. A preferred diet is diet I.Active Site Titration (AST, Example 6) is preferably be used todetermine the lipase concentration.

(C) In a still further first particular embodiment, the lipase of theinvention is selected from amongst lipases having the followingsubstitutions, preferably sets of substitutions, as compared to thelipase of SEQ ID NO: 2:

-   N33Q+E219D+T231R+N233R;-   N33Q+W117L+T231R+N233R;-   D27Q+N33Q+T231R+N233R;-   N33Q+G91T+T231R+N233R;-   D27S+N33Q+G91A+D96E+L97Q+D111A+S216P+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+D111A+T231R+N233R+P256T;-   D27R+N33Q+G91T+N94S+D111A+S216P+L227G+T231R+N233R+P256T;-   Q4R+N33Q+T231R+N233R;-   N33Q+T231R+N233R+Q249R;-   N33Q+D96W+T231R+N233R; and-   N33Q+G91N+T231R+N233R.

In a still further second particular embodiment, the lipase of theinvention is selected from amongst lipases having the followingsubstitutions, preferably sets of substitutions, as compared to thelipase of SEQ ID NO: 2:

-   N33Q+E219D+T231R+N233R;-   D27Q+N33Q+T231R+N233R;-   N33Q+G91T+T231R+N233R; and-   D27R+N33Q+G91T+N94S+D111A+S216P+L227G+T231R+N233R+P256T.

In a still further third particular embodiment, the lipase of theinvention is selected from amongst lipases having the followingsubstitutions, preferably sets of substitutions, as compared to thelipase of SEQ ID NO: 2:

-   N33Q+E219D+T231R+N233R; and-   N33Q+G91T+T231R+N233R.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the three still further particularembodiments) all have an improved in vitro digestion performance, i.e.,an improvement factor (IF), preferably average IF minus standarddeviation, of above 1.00, or at least 1.50 (or 1.5), 2.00 (or 2.0), 2.50(or 2.5), 3.00 (or 3.0), 3.50 (or 3.5), or at least 4.00 (or 4.0),preferably of at least 5.00 (or 5.0), 6.00 (or 6.0), 7.00 (or 7.0), 8.00(or 8.0), 9.00 (or 9.0), 10.00 (or 10.0), or at least 11.00 (or 11.0). Agastric step of pH 3 is preferably used. A preferred diet is diet II.Active Site Titration (AST, Example 6) is preferably used to determinethe lipase concentration.

(D) In a first particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+D167E+T231R+N233R;-   N33Q+E87A+T231R+N233R;-   N33Q+E210V+T231R+N233R;-   N33Q+E56K+T231R+N233R;-   N33Q+T231R+N233R+D254G;-   N33Q+D96S+T231R+N233R;-   N33Q+D122N+T231R+N233R;-   N26A+N33Q+T231R+N233R;-   N33Q+N162T+T231R+N233R;-   N33Q+A150V+N162G+T231R+N233R;-   N33Q+T231R+N233R+G240L;-   N33Q+E210V+T231R+N233R+D254S;-   N11R+N33Q+E210V+T231R+N233R+D254S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R;-   Q4R+D27R+N33Q+G91T+N94S+D111A+E210D+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+E219D+T231R+N233R;-   N33Q+G163R+T231R+N233R;-   N33Q+G163N+T231R+N233R;-   N33Q+G163C+T231R+N233R;-   N33Q+G163Q+T231R+N233R;-   N33Q+G163E+T231R+N233R;-   N33Q+G163H+T231R+N233R;-   N33Q+G163I+T231R+N233R;-   N33Q+G91K+T231R+N233R;-   N33Q+G91M+T231R+N233R;-   N33Q+G91F+T231R+N233R;-   N33Q+G91S+T231R+N233R;-   N33Q+G91W+T231R+N233R;-   N33Q+G91Y+T231R+N233R;-   N33Q+G163Y+T231R+N233R;-   N33Q+G163V+T231R+N233R;-   N33Q+G91C+T231R+N233R;-   N33Q+G91Y+Q126L+T231R+N233R;-   N33Q+G91M+G161E+T231R+N233R;-   N33Q+V128A+T231R+N233R;-   N33Q+V128A+T231R+N233R;-   N33Q+G163E+T231R+N233R;-   N33Q+G163V+L185M+T231R+N233R;-   N33Q+G38A+T231R+N233R;-   N33Q+G163A+T231R+N233R;-   N33Q+G91T+N94S+D111A+T231R+N233R;-   N33Q+G163M+T231R+N233R;-   N33Q+G91V+T231R+N233R;-   N33Q+D111A+T231R+N233R+Q249R;-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+D254G+P256T;-   N33Q+G91T+N94R+T231R+N233R+D254S;-   N33Q+G91T+N94R+D111A+W117L+T231R+N233R;-   N33Q+W117L+T231R+N233R+D254S;-   N33Q+T231R+N233R+P256T;-   N33Q+T231R+N233R+D242E;-   N33Q+E87R+T231R+N233R;-   N33Q+E56R+T231R+N233R;-   N33Q+N162G+T231R+N233R;-   N33Q+G91L+T231R+N233R;-   N33Q+E87H+T231R+N233R;-   N33Q+D96N+T231R+N233R+Q249R;-   N33Q+G91T+N94R+T231R+N233R+D254S;-   N33Q+L227F+T231R+N233R+D254S;-   N33Q+G163A+T231R+N233R;-   D27R+N33Q+G91T+D96E+D111A+T231R+N233R+D254S+P256T;-   N33Q+G91T+N94R+T231R+N233R;-   N33Q+T231R+N233R+D254A;-   N33Q+T231R+N233R+D254N;-   N33Q+T231R+N233R+D254L;-   N33Q+T231R+N233R+D254K;-   N33Q+T231R+N233R+D254M;-   D27V+N33Q+V60S+G91T+D96W+T231R+N233R+Q249R;-   N33Q+D96N+L227G+T231R+N233R+Q249R;-   D27R+N33Q+L227G+T231R+N233R;-   D27R+N33Q+L227G+T231R+N233R+Q249R;-   N33Q+E219D+L227G+T231R+N233R+Q249R;-   D27Q+N33Q+L227G+T231R+N233R+Q249R;-   N33Q+W117L+L227G+T231R+N233R+Q249R;-   D5E+N33Q+W117L+L227G+T231R+N233R+Q249R;-   D27Q+N33Q+E219D+L227G+T231R+N233R+Q249R;-   N33Q+D96E+E219D+L227G+T231R+N233R+Q249R;-   D27R+N33Q+E56K+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111A+S216P+L227G+T231R+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E56S+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91N+N94R+D111S+A155V+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   N33Q+D111A+T231R+N233R+D254S;-   N33Q+D111A+W117L+T231R+N233R+D254S;-   N33Q+T231R+N233R+P256N;-   N33Q+T231R+N233R+P256G;-   N33Q+T231R+N233R+P256H;-   N33Q+T231R+N233R+P256M;-   N33Q+T231R+N233R+P256W;-   N33Q+T231R+N233R+P256Y;-   N33Q+T231R+N233R+P256F;-   N33Q+T231R+N233R+P256V;-   N33Q+G91M+G163W+T231R+N233R;-   N33Q+G91M+G163T+T231R+N233R;-   N33Q+G91M+G163D+T231R+N233R;-   N33Q+G91K+G163W+T231R+N233R;-   N33Q+G91T+G163W+T231R+N233R;-   N33Q+V176N+T231R+N233R;-   N33Q+V176D+T231R+N233R;-   N33Q+W117F+T231R+N233R;-   N33Q+V176I+T231R+N233R;-   N33Q+D111N+T231R+N233R;-   N33Q+D111N+G225P+T231R+N233R;-   N33Q+D111N+S216P+T231R+N233R;-   D27R+N33Q+G91T+N94R+D111A+S216P+L227G+T231R+N233R;-   N33Q+G91M+G163P+T231R+N233R;-   N33Q+G91T+G163A+T231R+N233R;-   N33Q+W117D+T231R+N233R;-   N33Q+W117H+T231R+N233R;-   N33Q+W117C+T231R+N233R;-   N33Q+W117C+T231R+N233R;-   N33Q+W117K+T231R+N233R;-   N33Q+W117V+T231R+N233R;-   N11S+N33Q+T231R+N233R;-   N33Q+W117E+V176K+T231R+N233R;-   N33Q+W117G+T231R+N233R;-   N33Q+W117P+T231R+N233R;-   N33Q+W117S+T231R+N233R;-   N33Q+W117T+T231R+N233R;-   N33Q+W117I+T231R+N233R;-   D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;-   N33Q+V176M+T231R+N233R;-   N33Q+V176H+T231R+N233R;-   N33Q+V176A+T231R+N233R;-   D27V+N33Q+L227F+T231R+N233R+Q249R;-   N33Q+W117Y+T231R+N233R;-   N33Q+W117Y+V176D+T231R+N233R;-   D27R+N33Q+P136H+L227G+T231R+N233R+Q249R+D254S;-   N11R+N33Q+T231R+N233R+T244S;-   N33Q+G91T+D96N+D111A+V176I+T231R+N233R+D254S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;-   N33Q+G161A+T231R+N233R;-   N33Q+G38I+G177A+T231R+N233R;-   N33Q+N101Q+T231R+N233R;-   N33Q+N94Q+T231R+N233R;-   N11Q+N33Q+T231R+N233R;-   N8Q+N33Q+T231R+N233R;-   N33Q+T231R+N233R+N251Q;-   N33Q+N200Q+T231R+N233R;-   N33Q+G177A+T231R+N233R;-   N33Q+N73Q+T231R+N233R;-   N33Q+186L+T231R+N233R;-   N33Q+K98I+G163K+T231R+N233R;-   D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;-   D27R+N33Q+S216P+L227G+T231R+N233R+Q249R;-   N33Q+K98I+G163K+N200Q+T231R+N233R+N251S;-   N33Q+G38S+G163K+T231R+N233R;-   N33Q G38Y T231R N233R;-   D27R+N33Q+G91T+N94R+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91T+N94R+D111A+S216P+L227G+T231R+N233R+P256T;-   N33Q+G38N+N73Q+T231R+N233R;-   N33Q+G38D+R84E+T231R+N233R;-   N33Q+G38Q+T231R+N233R;-   N33Q+G38I+T231R+N233R;-   N33Q+G38K+T231R+N233R;-   N33Q+G38F+T231R+N233R;-   N33Q+G38H+N200Q+T231R+N233R+N251S;-   N33Q+G38L+T231R+N233R;-   N33Q+G38P+T231R+N233R;-   N33Q+G38T+T231R+N233R;-   N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;-   D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;-   D27R+N33Q+G38A+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;-   D27R+N33Q+V176Q+L227G+T231R+N233R+Q249R+D254S;-   N33Q+W117I+V176Q+T231R+N233R+P256A;-   N33Q+G38A+G163A+T231R+N233R+P256A;-   N33Q+W117I+V176Q+T231R+N233R;-   N33Q+G177A+T231R+N233R+G246A;-   E1N N33Q T231R N233R;-   N33Q G38H T231R N233R;-   N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;-   N33Q+K98I+T231R+N233R;-   D27R+N33Q+W117I+V176Q+L227G+T231R+N233R+Q249R+D254S;-   N11R+N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   N33Q+G163W+T231R+N233R;-   N33Q+G38A+G163A+T231R+N233R;-   D27R+N33Q+G91T+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;-   N33Q+T231R+N233R+D254Q;-   N11R+N33Q+G91T+S115L+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+V176W+T231R+N233R+D254S;-   N33Q+G163D+T231R+N233R;-   N33Q+G163D+T231R+N233R;-   N33Q+G163P+T231R+N233R;-   E1D+N33Q+G91T+N94R+D111A+W117L+T231R+N233R+D254S;-   N33Q+G91T+N94R+D111A+W117L+V176W+T231R+N233R;-   Q4P+D27R+N33Q+G91N+N94R+D111A+L206F+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+T37K+N71I+G91N+N94R+K98I+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   N33Q+W117S+T231R+N233R;-   N33Q+G61R+V63R+G156R+V176W+T231R+N233R+P256I;-   N33Q+D96N+G156R+V176W+T231R+N233R;-   N33Q+G156R+V176W+T231R+N233R+Q249R;-   N33Q+G91T+N94S+D111A+G163T+V176W+T231R+N233R;-   N33Q+G91T+N94S+D111A+S115L+G163T+V176I+T231R+N233R;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+D254S+P256T;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D242E+D254S+P256T;-   D27R+N33Q+G38A+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+E210D+S216P+L227G+T231R+N233R+P256L;-   N33Q+G38A+G91T+G163A+T231R+N233R+D254S;-   N33Q+G38A+G163A+T231R+N233R+D254I;-   N11R+N33Q+I90L+G163L+T231R+N233R;-   N11R+N33Q+I90L+G163L+T231R+N233R+D254S;-   N11R+N33Q+E56Q+G91T+G163K+V176Q+T231R+N233R+D254S;-   N11R+D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+G12A+G163A+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+E210D+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254I;-   N11R+N33Q+G91T+G163K+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+T231R+N233R+D254S;-   N11R+N33Q+G91M+G163T+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163K+V176D+T231R+N233R+D254S;-   N33Q+E56Q+G156R+V176W+T231R+N233R;-   E1D+N33Q+G38A+G91T+N94R+D111A+W117L+V176W+T231R+N233R;-   N33Q+G163K+G177A+T231R+N233R+G246A;-   N11R+N33Q+E56Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+190L+G163K+T231R+N233R+D254S;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+E219D+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+I90L+G91T+N94S+D96E+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163A+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S+P256N;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+G163A+E210V+S216P+L227G+T231R+N233R+P256L;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+V176I+E210V+S216P+L227G+T231R+N233R+P256L;-   N33Q+E210Y+T231R+N233R+D254Y+I255F;-   N33Q+L93F+D102Y+T231R+N233R;-   D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;-   N11S+N33Q+T231R+N233R;-   N11R+N33Q+T231R+N233R:-   N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   N33Q+W117Y+V176T+T231R+N233R;-   N8L+N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   N11R+N33Q+G38A+G91T+G163P+V176G+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254A+P256F;-   N11R+N33Q+G91T+G163K+T231R+N233R+P256F;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256F;-   N11R+N33Q+G38A+G91T+G156R+G163K+V176T+T231R+N233R+D254S;-   N33Q+G91K+D96S+G163T+T231R+N233R+Q249R;-   N11R+N33Q+G91T+G163N+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163W+T231R+N233R+D254S:-   N11R+N33Q+G91K+G163K+T231R+N233R+D254S;-   N11R+G23E+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+V141E+G163K+T231R+N233R+D254S;-   N11R+N33Q+L52R+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+V141L+G163K+T231R+N233R+D254S;-   N11R+N33Q+T37K+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+A68V+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163A+V176I+T231R+N233R+D254S;-   N11R+N33Q+T37M+G91T+G163P+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256I;-   N33Q+G38S+G156R+G163K+V176W+T231R+N233R;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163K+S216P+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+G163P+V176G+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163Q+V176G+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163T+V176G+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+N94R+G163P+V176G+T231R+N233R+D254S;-   E1*+N11R+N33Q+G38A+G91N+N94R+G163P+V176G+T231R+N233R+D254S;-   E1N+N11R+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   E1N+F10L+N11R+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S;-   E1N+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+L227F+T231R+N233R;-   E1N+N11R+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+L227F+T231R+N233R+D254S;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S+I255A+P256Q;-   E1N+N11R+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R+D254S;-   N33Q+G156R+V176W+T231R+N233R+P256I;-   N33Q+G91T+N94S+D111A+G156R+G163T+V176W+T231R+N233R;-   N33Q+G91T+N94S+D111A+G156R+G163T+V176I+T231R+N233R;-   N11R+N33Q+G38A+G91T+D102G+S115L+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+S115L+G163K+T231R+N233R+D254S+P256T;-   E1N+N11R+N33Q+G91T+G163A+T231R+N233R+G246A+D254S;-   N11R+D27R+N33Q+D57G+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N33Q+D96N+G156R+V176W+T231R+N233R+Q249R;-   N33Q+I86F+L93F+D102Y+E210Y+L227F+T231R+N233R+D254Y+I255F+L269F;-   N33Q+I86F+L93F+D102Y+E210Y+L227F+T231R+N233R+D254Y+I255F:-   N11C+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11L+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11H+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11D+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+D96W+G163K+T231R+N233R+D254S;-   D27R+N33Q+G91T+D96E+L97Q+D111A+G163K+T231R+N233R+D254S+P256T;-   N11P+N33Q+G91T+G163K+T231R+N233R+D254S;-   Q4R+D27N+N33Q+G38A+G91T+N94S+E99D+D111A+V176I+E210V+S216P+L227G+T231R+N233R+P256L;-   N11R+N33Q+E56Q+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163A+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+T231R+N233R+D254S; and-   N11R+N33Q+G91T+G163K+L227G+P229R+T231R+N233R+D254S.

In a second particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+E87A+T231R+N233R;-   N33Q+E210V+T231R+N233R;-   N33Q+E56K+T231R+N233R;-   N33Q+T231R+N233R+D254G;-   N33Q+D96S+T231R+N233R;-   N33Q+N162T+T231R+N233R;-   N33Q+T231R+N233R+G240L;-   N33Q+E210V+T231R+N233R+D254S;-   N11R+N33Q+E210V+T231R+N233R+D254S;-   N33Q+G163N+T231R+N233R;-   N33Q+G163Q+T231R+N233R;-   N33Q+G163E+T231R+N233R;-   N33Q+G91M+T231R+N233R;-   N33Q+G91F+T231R+N233R;-   N33Q+G163Y+T231R+N233R;-   N33Q+G91Y+Q126L+T231R+N233R;-   N33Q+V128A+T231R+N233R;-   N33Q+G38A+T231R+N233R;-   N33Q+G163M+T231R+N233R;-   N33Q+D111A+T231R+N233R+Q249R;-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+D254G+P256T;-   N33Q+G91T+N94R+T231R+N233R+D254S;-   N33Q+G91T+N94R+D111A+W117L+T231R+N233R;-   N33Q+W117L+T231R+N233R+D254S;-   N33Q+T231R+N233R+P256T;-   N33Q+T231R+N233R+D242E;-   N33Q+E87R+T231R+N233R;-   N33Q+E56R+T231R+N233R;-   N33Q+N162G+T231R+N233R;-   N33Q+G91L+T231R+N233R;-   N33Q+E87H+T231R+N233R;-   N33Q+D96N+T231R+N233R+Q249R;-   N33Q+G91T+N94R+T231R+N233R+D254S;-   N33Q+L227F+T231R+N233R+D254S;-   N33Q+G91T+N94R+T231R+N233R;-   N33Q+T231R+N233R+D254L;-   N33Q+T231R+N233R+D254K;-   N33Q+T231R+N233R+D254M;-   D27V+N33Q+V60S+G91T+D96W+T231R+N233R+Q249R;-   N33Q+D96N+L227G+T231R+N233R+Q249R;-   N33Q+E219D+L227G+T231R+N233R+Q249R;-   D27Q+N33Q+E219D+L227G+T231R+N233R+Q249R;-   N33Q+D96E+E219D+L227G+T231R+N233R+Q249R;-   D27R+N33Q+E56K+G91N+N94R+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   N33Q+D111A+T231R+N233R+D254S;-   N33Q+T231R+N233R+P256N;-   N33Q+T231R+N233R+P256G;-   N33Q+T231R+N233R+P256M;-   N33Q+T231R+N233R+P256W;-   N33Q+W117F+T231R+N233R;-   N33Q+D111N+G225P+T231R+N233R;-   D27R+N33Q+G91T+N94R+D111A+S216P+L227G+T231R+N233R;-   N33Q+W117C+T231R+N233R;-   N33Q+W117C+T231R+N233R;-   N33Q+W117K+T231R+N233R;-   N11S+N33Q+T231R+N233R;-   N33Q+W117G+T231R+N233R;-   N33Q+W117P+T231R+N233R;-   N33Q+W117S+T231R+N233R;-   N33Q+W 17T+T231R+N233R;-   D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;-   N11R+N33Q+T231R+N233R+T244S;-   N33Q+G91T+D96N+D111A+V176I+T231R+N233R+D254S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;-   N33Q+G38I+G177A+T231R+N233R;-   N33Q+N101Q+T231R+N233R;-   N33Q+N94Q+T231R+N233R;-   N11Q+N33Q+T231R+N233R;-   N33Q+G177A+T231R+N233R;-   N33Q+N73Q+T231R+N233R;-   D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;-   N33Q+G38I+T231R+N233R;-   N33Q+G38F+T231R+N233R;-   N33Q+G38H+N200Q+T231R+N233R+N251S;-   N33Q+G38T+T231R+N233R;-   N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;-   D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;-   D27R+N33Q+G38A+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;-   D27R+N33Q+V176Q+L227G+T231R+N233R+Q249R+D254S;-   N33Q+G38A+G163A+T231R+N233R+P256A;-   N33Q+W117I+V176Q+T231R+N233R;-   N33Q+G177A+T231R+N233R+G246A;-   N33Q G38H T231R N233R;-   N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   N33Q+G163W+T231R+N233R;-   N33Q+G38A+G163A+T231R+N233R;-   D27R+N33Q+G91T+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;-   N33Q+G91T+N94R+D111A+W117L+V176W+T231R+N233R;-   Q4P+D27R+N33Q+G91N+N94R+D111A+L206F+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+190V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   N33Q+W117S+T231R+N233R;-   N33Q+G61R+V63R+G156R+V176W+T231R+N233R+P256I;-   N33Q+D96N+G156R+V176W+T231R+N233R;-   N33Q+G91T+N94S+D111A+S115L+G163T+V176I+T231R+N233R;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D242E+D254S+P256T;-   D27R+N33Q+G38A+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+I90L+G163L+T231R+N233R;-   N11R+D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+G112A+G163A+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+E210D+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254I;-   N11R+N33Q+G91T+G163P+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163K+V176D+T231R+N233R+D254S;-   E1D+N33Q+G38A+G91T+N94R+D111A+W117L+V176W+T231R+N233R;-   N33Q+G163K+G177A+T231R+N233R+G246A;-   N11R+N33Q+E56Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+190L+G163K+T231R+N233R+D254S;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+E219D+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+G91T+G163K+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163A+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S+P256N;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+G163A+E210V+S216P+L227G+T231R+N233R+P256L;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+V176I+E210V+S216P+L227G+T231R+N233R+P256L;-   D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;-   N11R+N33Q+T231R+N233R;-   N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   N33Q+W117Y+V176T+T231R+N233R;-   N8L+N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163P+V176G+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254A+P256F;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256F;-   N11R+N33Q+G91T+G163N+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163W+T231R+N233R+D254S;-   N11R+N33Q+G91K+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+V141E+G163K+T231R+N233R+D254S;-   N11R+N33Q+L52R+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+T37K+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+A68V+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163A+V176I+T231R+N233R+D254S;-   N11R+N33Q+T37M+G91T+G163P+V176T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256I;-   N33Q+G38S+G156R+G163K+V176W+T231R+N233R;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163K+S216P+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+G163Q+V176G+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+G163T+V176G+T231R+N233R+D254S;-   N11R+N33Q+G38A+G91T+N94R+G163P+V176G+T231R+N233R+D254S;-   E1N+N11R+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   E1N+F10L+N11R+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S;-   E1N+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+L227F+T231R+N233R;-   E1N+N11R+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+L227F+T231R+N233R+D254S;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S+I255A+P256Q;-   E1N+N11R+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R+D254S;-   N33Q+G156R+V176W+T231R+N233R+P256I;-   N11R+N33Q+G38A+G91T+D102G+S115L+G163K+T231R+N233R+D254S+256T;-   N11R+N33Q+G38A+G91T+S115L+G163K+T231R+N233R+D254S+P256T;-   E1N+N11R+N33Q+G91T+G163A+T231R+N233R+G246A+D254S;-   N11R+D27R+N33Q+D57G+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N33Q+D96N+G156R+V176W+T231R+N233R+Q249R;-   N11C+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11L+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11H+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11D+N33Q+G91T+G163K+T231R+N233R+D254S,-   N11R+N33Q+G91T+D96W+G163K+T231R+N233R+D254S;-   D27R+N33Q+G91T+D96E+L97Q+D111A+G163K+T231R+N233R+D254S+P256T;-   N11P+N33Q+G91T+G163K+T231R+N233R+D254S;-   Q4R+D27N+N33Q+G38A+G91T+N94S+E99D+D111A+V176I+E210V+S216P+L227G+T231R+N233R+P256L;-   N11R+N33Q+E56Q+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163A+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+T231R+N233R+D254S; and-   N11R+N33Q+G91T+G163K+L227G+P229R+T231R+N233R+D254S.

In a third particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+E87A+T231R+N233R;-   N33Q+T231R+N233R+D254G;-   N33Q+T231R+N233R+G240L;-   N33Q+E210V+T231R+N233R+D254S;-   N11R+N33Q+E210V+T231R+N233R+D254S;-   N33Q+G91Y+Q126L+T231R+N233R;-   N33Q+G163M+T231R+N233R;-   N33Q+D111A+T231R+N233R+Q249R;-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+D254G+P256T;-   N33Q+G91T+N94R+T231R+N233R+D254S;-   N33Q+W117L+T231R+N233R+D254S;-   N33Q+T231R+N233R+D242E;-   N33Q+E87R+T231R+N233R;-   N33Q+E56R+T231R+N233R;-   N33Q+N162G+T231R+N233R;-   N33Q+T231R+N233R+D254L;-   N33Q+T231R+N233R+D254K;-   D27V+N33Q+V60S+G91T+D96W+T231R+N233R+Q249R;-   N33Q+D96N+L227G+T231R+N233R+Q249R;-   N33Q+E219D+L227G+T231R+N233R+Q249R;-   D27Q+N33Q+E219D+L227G+T231R+N233R+Q249R;-   N33Q+D96E+E219D+L227G+T231R+N233R+Q249R;-   N33Q+D111A+T231R+N233R+D254S;-   N33Q+T231R+N233R+P256N;-   N33Q+T231R+N233R+P256M;-   D27R+N33Q+G91T+N94R+D111A+S216P+L227G+T231R+N233R;-   D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;-   N33Q+G91T+D96N+D111A+V176I+T231R+N233R+D254S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;-   N33Q+G38I+G177A+T231R+N233R;-   N33Q+G177A+T231R+N233R;-   D27R+N33Q+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;-   N33Q+G38F+T231R+N233R;-   N33Q+G38H+N200Q+T231R+N233R+N251S;-   N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;-   D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;-   D27R+N33Q+V176Q+L227G+T231R+N233R+Q249R+D254S;-   N33Q+G38A+G163A+T231R+N233R+P256A;-   N33Q+W117I+V176Q+T231R+N233R;-   N33Q+G91T+N94R+D111A+W117L+V176W+T231R+N233R;-   Q4P+D27R+N33Q+G91N+N94R+D111A+L206F+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   N33Q+D96N+G156R+V176W+T231R+N233R;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D242E+D254S+P256T;-   D27R+N33Q+G38A+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+190L+G163L+T231R+N233R;-   N11R+D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+G112A+G163A+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254I;-   N11R+N33Q+G38A+G91T+G163K+V176D+T231R+N233R+D254S;-   N33Q+G163K+G177A+T231R+N233R+G246A;-   D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;-   N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+V176I+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163L+T231R+N233R+D254S+P256N;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+G163A+E210V+S216P+L227G+T231R+N233R+P256L;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+V176I+E210V+S216P+L227G+T231R+N233R+P256L;-   N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254A+P256F;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256F;-   N11R+N33Q+G91T+G163T+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163W+T231R+N233R+D254S;-   N11R+N33Q+G91K+G163K+T231R+N233R+D254S;-   N11R+N33Q+L52R+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+T37K+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+A68V+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+T37M+G91T+G163P+V176T+T231R+N233R+D254S;-   N33Q+G38S+G156R+G163K+V176W+T231R+N233R;-   N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163K+S216P+L227G+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+G163T+V176G+T231R+N233R+D254S;-   E1N+N11R+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   E1N+F10L+N11R+N33Q+G38A+G91T+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S;-   E1N+N11R+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R;-   E1N+N33Q+G38A+G91T+G163P+V176F+L227F+T231R+N233R+D254S;-   E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S+I255A+P256Q;-   N11R+N33Q+G38A+G91T+D102G+S115L+G163K+T231R+N233R+D254S+P256T;-   N11R+N33Q+G38A+G91T+S115L+G163K+T231R+N233R+D254S+P256T;-   E1N+N11R+N33Q+G91T+G163A+T231R+N233R+G246A+D254S;-   N11R+D27R+N33Q+D57G+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N33Q+D96N+G156R+V176W+T231R+N233R+Q249R;-   N11C+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11L+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11H+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11D+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+D96W+G163K+T231R+N233R+D254S;-   D27R+N33Q+G91T+D96E+L97Q+D111A+G163K+T231R+N233R+D254S+P256T;-   N11P+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+E56Q+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163P+T231R+N233R+D254S; and-   N11R+N33Q+G91T+G163K+L227G+P229R+T231R+N233R+D254S.

In a fourth particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+G91T+N94R+T231R+N233R+D254S;-   N33Q+E87R+T231R+N233R;-   D27V+N33Q+V60S+G91T+D96W+T231R+N233R+Q249R;-   N33Q+E219D+L227G+T231R+N233R+Q249R;-   N33Q+D96E+E219D+L227G+T231R+N233R+Q249R;-   D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;-   D27R+N33Q+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;-   D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;-   N33Q+G38A+G163A+T231R+N233R+P256A;-   N11R+N33Q+190L+G163L+T231R+N233R;-   N11R+N33Q+G38A+G91T+G112A+G163A+T231R+N233R+D254S;-   Q4R+D27Q+N33Q+G91T+N94S+E99D+D111A+V176I+E210V+S216P+L227G+T231R+N233R+P256L;-   N11R+N33Q+G91T+G163W+T231R+N233R+D254S;-   N11R+N33Q+L52R+G91T+G163K+T231R+N233R+D254S;-   E1N+N11R+N33Q+G91T+G163A+T231R+N233R+G246A+D254S;-   N33Q+D96N+G156R+V176W+T231R+N233R+Q249R;-   N11L+N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+D96W+G163K+T231R+N233R+D254S; and-   D27R+N33Q+G91T+D96E+L97Q+D111A+G163K+T231R+N233R+D254S+P256T.

In a fifth particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N11R+N33Q+G91T+G163W+T231R+N233R+D254S;-   N33Q+G38S+G156R+G163K+V176W+T231R+N233R; and-   N11L+N33Q+G91T+G163K+T231R+N233R+D254S.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the five particular embodiments) allhave an improved in vitro digestion performance, i.e., an improvementfactor (IF), preferably average IF minus standard deviation, of above1.00, or of at least 1.50 (or 1.5), 2.00 (or 2.0), 2.50 (or 2.5), 3.00(or 3.0), 3.50 (or 3.5), or at least 4.00 (or 4.0), preferably of atleast 5.00 (or 5.0), 6.00 (or 6.0), 7.00 (or 7.0), 8.00 (or 8.0), 9.00(or 9.0), 10.00 (or 10.0), or at least 11.00 (or 11.0). A gastric stepof pH 5 is preferably used. A preferred diet is diet 1. Active SiteTitration (AST, Example 6) is preferably used to determine the lipaseconcentration.

(E) In a first particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+P256T;-   N33Q+E210D+T231R+N233R;-   N33Q+T231R+N233R;-   N33Q+D111A+T231R+N233R;-   N33Q+G91T+T231R+N233R;-   D27Q+N33Q+T231R+N233R;-   Q9H+N33Q+D102E+T231R+N233R;-   N33Q+E56Q+T231R+N233R;-   N33Q+190L+G163L+T231R+N233R;-   D27R+N33Q+G91A+D96E+D111A+T231R+N233R+D254G+P256T;-   N33Q+N39S+T231R+N233R;-   N33Q+N94R+T231R+N233R;-   N33Q+T231R+N233R+D254S;-   N33Q+G91T+G163K+T231R+N233R+D254G;-   N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S;-   N33Q+T231R+N233R;-   K98I+T231R+N233R+N251S;-   N33Q+G163P+T231R+N233R;-   N33Q+G163D+T231R+N233R;-   N33Q+G163T+T231R+N233R;-   N33Q+G163W+T231R+N233R;-   N33Q+G38A+G163A+T231R+N233R;-   N33Q+D11A+T231R+N233R+D254S;-   D27R+N33Q+G91T+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;-   N33Q+T231R+N233R+P256A;-   N33Q+T231R+N233R+P256S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;-   N33Q+S115L+T231R+N233R;-   N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   D27R+N33Q+G38A+G91T+D96E+D111A+T231R+N233R+D254S+P256T;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S;-   N33Q+G91T+K98I+G163K+T231R+N233R+D254S+P256L; and-   N33Q+G91T+T114I+G163K+T231R+N233R+D254S+P256L.

In a second particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+P256T;-   N33Q+E210D+T231R+N233R;-   N33Q+T231R+N233R;-   N33Q+D111A+T231R+N233R;-   N33Q+G91T+T231R+N233R;-   N33Q+N94R+T231R+N233R;-   N33Q+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S;-   N33Q+G163P+T231R+N233R;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S; and-   N33Q+G91T+T114I+G163K+T231R+N233R+D254S+P256L.

In a third particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+P256T;-   N33Q+G91T+T231R+N233R;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S; and-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the three particular embodiments)all have an improved in vitro digestion performance, i.e., animprovement factor (IF), preferably average IF minus standard deviation,of above 1.00, or of at least 1.50 (or 1.5), 2.00 (or 2.0), 2.50 (or2.5), 3.00 (or 3.0), 3.50 (or 3.5), or at least 4.00 (or 4.0),preferably of at least 5.00 (or 5.0), 6.00 (or 6.0), 7.00 (or 7.0), 8.00(or 8.0), 9.00 (or 9.0), 10.00 (or 10.0), or at least 11.00 (or 11.0). Agastric step of pH 3 is preferably used. A preferred diet is diet I.Active Site Titration (AST, Example 6) is preferably used to determinethe lipase concentration.

(F) In a first particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+P256T;-   D27R+N33Q+G91A+D96E+L97Q+D1111A+T231R+N233R+P256T;-   N33Q+E210D+T231R+N233R;-   N33Q+D111A+T231R+N233R;-   N33Q+G91T+T231R+N233R;-   D27Q+N33Q+T231R+N233R;-   Q9H+N33Q+D102E+T231R+N233R;-   N33Q+G91T+G163K+T231R+N233R;-   N33Q+E56Q+T231R+N233R;-   N33Q+190L+G163L+T231R+N233R;-   N33Q+N94R+T231R+N233R;-   N33Q+T231R+N233R+D254S;-   N33Q+G91T+G163K+T231R+N233R+D254G;-   N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S;-   N33Q+T231R+N233R;-   K98I+T231R+N233R+N251S;-   N33Q+G163P+T231R+N233R;-   N33Q+G163D+T231R+N233R;-   N33Q+G163T+T231R+N233R;-   N33Q+G163W+T231R+N233R;-   N33Q+G38A+G163A+T231R+N233R;-   N33Q+D111A+T231R+N233R+D254S;-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;-   D27R+N33Q+G91T+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;-   N33Q+T231R+N233R+D254Q;-   N33Q+T231R+N233R+D254I;-   N33Q+S216P+L227G+T231R+N233R+Q249R;-   N33Q+T231R+N233R+P256A;-   N33Q+T231R+N233R+P256L;-   N33Q+T231R+N233R+P256S;-   N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;-   N33Q+S115L+T231R+N233R;-   N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R;-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   N33Q+G161A+T231R+N233R;-   N33Q G38M T231R N233R;-   N33Q G38F T231R N233R;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S;-   N33Q+G91T+K98I+G163K+T231R+N233R+D254S+P256L; and-   N33Q+G91T+T114I+G163K+T231R+N233R+D254S+P256L.

In a second particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+P256T;-   D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+P256T;-   N33Q+E210D+T231R+N233R;-   N33Q+D111A+T231R+N233R;-   N33Q+G91T+T231R+N233R;-   D27Q+N33Q+T231R+N233R;-   N33Q+E56Q+T231R+N233R;-   N33Q+I90L+G163L+T231R+N233R;-   N33Q+G91T+G163K+T231R+N233R+D254G;-   N33Q+G91T+G163K+T231R+N233R+D254S;-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S;-   N33Q+G163P+T231R+N233R;-   N33Q+G163D+T231R+N233R;-   N33Q+G163W+T231R+N233R;-   N33Q+G38A+G163A+T231R+N233R;-   N33Q+T231R+N233R+D254Q;-   N33Q+T231R+N233R+D254I;-   N33Q+T231R+N233R+P256S;-   N33Q+S115L+T231R+N233R;-   N33Q+G38A+G91T+G163K+T231R+N233R+D254S;-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R;-   N33Q G38M T231R N233R;-   N33Q G38F T231R N233R;-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S;-   N33Q+G91T+K98I+G163K+T231R+N233R+D254S+P256L; and-   N33Q+G91T+T114I+G163K+T231R+N233R+D254S+P256L.

In a third particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+E210D+T231R+N233R;-   N33Q+G91T+T231R+N233R;-   D27Q+N33Q+T231R+N233R;-   N33Q+190L+G163L+T231R+N233R;-   D27N+N33Q+G91T+G163K+T231R+N233R+D254S;-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R;-   N33Q G38M T231R N233R;-   N33Q G38F T231R N233R;-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S;

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the three particular embodiments)all have an improved in vitro digestion performance, i.e., animprovement factor (IF), preferably average IF minus standard deviation,of above 1.00, or of at least 1.50 (or 1.5), 2.00 (or 2.0), 2.50 (or2.5), 3.00 (or 3.0), 3.50 (or 3.5), or at least 4.00 (or 4.0),preferably of at least 5.00 (or 5.0), 6.00 (or 6.0), 7.00 (or 7.0), 8.00(or 8.0), 9.00 (or 9.0), 10.00 (or 10.0), or at least 11.00 (or 11.0). Agastric step of pH 5 is preferably used. A preferred diet is diet I.A₂₈₀ is preferably used to determine the lipase concentration,preferably using the extinction coefficient 1.24 A₂₈₀/mg.

The lipases of the invention may have a bile salt ratio improvement vsreference of at least 1.2, 1.4, 1.6, 1.8, or at least 2.0. Morepreferably the lipases of the invention may have a bile salt ratioimprovement vs reference of at least 2.2, 2.5, 2.8, or at least 3.0.Even more preferably the lipases of the invention may have a bile saltratio improvement vs reference of at least 3.2, 3.4, 3.6, 3.8, or atleast 4.0. These ratios may also be referred to as, e.g., 3×, 3-fold, or300%, all corresponding to a ratio of 3.0—and vice versa for otherratios.

In a particular embodiment, the lipase of the invention is selected fromamongst lipases having the following substitutions, preferably sets ofsubstitutions, as compared to the lipase of SEQ ID NO: 2:

-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   N33Q+G38W+G91T+T114I+G163K+E210D+T231R+N233R+P256T;-   D27I+N33Q+G91T+D96E+K98T+T114I+G163K+E210D+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+T231R+N233R+G163S;-   N33Q+G38W+G91T+T114I+G163K+E210V+T231R+N233R;-   Q4P+D27R+N33Q+G91N+N94R+D111A+R205I+L206F+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+G163K+E210D+T231R+N233R;-   D27R+N33Q+T37K+N71I+G91N+N94R+K98I+D111A+S216P+L227G+T231R+N233R+P256T;-   Q4H+D27R+N33Q+G91N+N94R+D111A+V154L+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+G163S+E210V+T231R+N233R+D254K+P256A;-   N33Q+G91T+T114I+G163K+E210D+T231R+N233R+D254G+P256A;-   D27R+N33Q+L52I+V60E+G91N+N94R+D111A+T114I+V168M+E210D+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+D111A+T114I+R179T+S216P+L227G+T231R+N233R+P256T;-   D27R+A30V+N33Q+G91N+N94R+G109A+D111A+G190D+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+K98I+D111A+N162S+S216P+L227G+T231R+N233R+P256T;-   N26H+D27R+N33Q+G91N+N94R+D111A+V154F+G190C+S216P+L227G+T231R+N233R+P256T;-   D27N+N33Q+G91T+T114I+G163S+E210D+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R;-   D27R+N33Q+T37K+N71I+G91N+N94R+K98I+D111A+S216P+L227G+Y231R+N233R+P256T;-   D27R+N33Q+G91T+T114I+G163W+E210D+T231R+N233R;-   D27R+N33Q+G91N+N94R+K98I+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+L97M+D111A+S216P+T226N+L227G+T231R+N233R+P256T+L269H;-   D27R+N33Q+G91N+N94R+D111A+V154I+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+T114I+E210V+T231R+N233R+D254K+P256A-   D27R+N33Q+N71S+G91N+N94R+D111A+H135D+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+T114I+G163K+E210D+T231R+N233R;-   D27R+N33Q+I76T+G91N+N94R+R108M+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+N39S+G91N+N94R+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+A49T+G91N+N94R+D111A+Y138F+G163R+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R;-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S; and-   N33Q+G91T+K98I+G163K+T231R+N233R+D254S+P256L.

In a more preferred embodiment, the lipase of the invention is selectedfrom amongst lipases having the following substitutions, preferably setsof substitutions, as compared to the lipase of SEQ ID NO: 2:

-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   N33Q+G38W+G91T+T114I+G163K+E210D+T231R+N233R+P256T;-   D27I+N33Q+G91T+D96E+K98T+T114I+G163K+E210D+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+T231R+N233R+G163S;-   N33Q+G38W+G91T+T114I+G163K+E210V+T231R+N233R;-   Q4P+D27R+N33Q+G91N+N94R+D111A+R205I+L206F+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+G163K+E210D+T231R+N233R;-   D27R+N33Q+T37K+N71I+G91N+N94R+K98I+D111A+S216P+L227G+T231R+N233R+P256T;-   Q4H+D27R+N33Q+G91N+N94R+D111A+V154L+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+G163S+E210V+T231R+N233R+D254K+P256A;-   N33Q+G91T+T114I+G163K+E210D+T231R+N233R+D254G+P256A;-   D27R+N33Q+L52I+V60E+G91N+N94R+D111A+T114I+V168M+E210D+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+D111A+T114I+R179T+S216P+L227G+T231R+N233R+P256T;-   D27R+A30V+N33Q+G91N+N94R+G109A+D111A+G190D+S216P+L227G+T231R+-   N233R+P256T;-   D27R+N33Q+G91N+N94R+K98I+D111A+N162S+S216P+L227G+T231R+N233R+P256T;-   N26H+D27R+N33Q+G91N+N94R+D111A+V154F+G190C+S216P+L227G+T231R+N233R+P256T;-   D27N+N33Q+G91T+T114I+G163S+E210D+T231R+N233R+P256T;-   D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R;-   D27R+N33Q+T37K+N71I+G91N+N94R+K98I+D111A+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+G91T+T114I+G163W+E210D+T231R+N233R;-   D27R+N33Q+G91N+N94R+K98I+D111A+S216P+L227G+T231R+N233R+D254S; and-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S; and-   N33Q+G91T+K98I+G163K+T231R+N233R+D254S+P256L.

In a most preferred embodiment, the lipase of the invention is selectedfrom amongst lipases having the following substitutions, preferably setsof substitutions, as compared to the lipase of SEQ ID NO: 2:

-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   N33Q+G38W+G91T+T114I+G163K+E210D+T231R+N233R+P256T;-   D27I+N33Q+G91T+D96E+K98T+T141I+G163K+E210D+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+T231R+N233R+G163S;-   N33Q+G38W+G91T+T114I+G163K+E210V+T231R+N233R;-   Q4P+D27R+N33Q+G91N+N94R+D111A+R205I+L206F+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+D96E+K98T+T114I+G163K+E210D+T231R+N233R; and-   N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the three embodiments) all have animproved in vitro digestion performance, i.e., an improved stability atpH 3 in the presence of pepsin, more in particular an improved residualactivity measured on PNP-palmitate at pH 8.0 and 20° C. (or roomtemperature) after incubation for 3 hours at pH 3.0 and 20° C. (or roomtemperature) in the presence of 75 ug/mL pepsin, preferably an improved% residual activity (% RA) as determined by the method of Example 8. Inparticular embodiments, the % RA is at least 30, at least 50, at least70, at least 80, or at least 90%. An improvement ratio may be defined asthe ratio of % RA of the lipase in question to the % RA of the referencelipase. This improvement ratio preferably is at least 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, or at least 4.5. The improvement ratio can becalculated from the results in Table 10 by dividing the % RA of thelipase in question with the % RA of the reference lipase (e.g., thelipase of SEQ ID NO: 2, or SEQ ID NO: 1, or another reference lipase, asdesired).

The lipases of the invention may have a bile salt ratio improvement vsreference of at least 1.2, 1.4, 1.6, 1.8, or at least 2.0. Morepreferably the lipases of the invention may have a bile salt ratioimprovement vs reference of at least 2.2, 2.5, 2.8, or at least 3.0.Even more preferably the lipases of the invention may have a bile saltratio improvement vs reference of at least 3.2, 3.4, 3.6, 3.8, or atleast 4.0. These ratios may also be referred to as, e.g., 3×, 3-fold, or300%, all corresponding to a ratio of 3.0— and vice versa for otherratios.

In a particular embodiment, the lipase of the invention is selected fromamongst lipases having the following substitutions, preferably sets ofsubstitutions, as compared to the lipase of SEQ ID NO: 2:

-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R+D254S:-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+190V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T-   D27R+N33Q+E43K+K46M+190V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   N33Q+G91T+T114I+E210V+D254K+P256A;-   N33Q+G91T+D96E+K98T+T114I+G163S+E210V+D254K+P256A;-   L52I+V60E+T114I+V168M+E210D;-   D27R+N33Q+A49T+G91N+N94R+D111A+Y138F+G163R+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+I76T+G91N+N94R+R108M+D111A+S216P+L227G+T231R+N233R+P256T;    and-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+P227G+T231R+N233R+P256T.

More preferably, the lipase of the invention is selected from amongstlipases having the following substitutions, preferably sets ofsubstitutions, as compared to the lipase of SEQ ID NO: 2:

-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;    and-   N33Q+G91T+T114I+E210V+D254K+P256A.

Even more preferably, the lipase of the invention is selected fromamongst lipases having the following substitutions, preferably sets ofsubstitutions, as compared to the lipase of SEQ ID NO: 2:

-   D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;-   N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;-   D27R+N33Q+E43K+K46M+I90V+G91N+N94R+D111A+T114I+S216P+L227G+T231R+N233R+P256T;-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T;    and-   N33Q+G91T+T114I+E210V+D254K+P256A.

In a most preferred embodiment, the lipase of the invention is selectedfrom amongst lipases having the following substitutions, preferably setsof substitutions, as compared to the lipase of SEQ ID NO: 2:

-   G23E+D27R+N33Q+L52R+G91N+N94R+D111A+T114I+V141E+S216P+L227G+T231R+N233R+P256T.

Variants of the lipase of amino acids 1-269 of SEQ ID NO: 2 with theabove-listed substitutions (each of the four embodiments) all have animproved in vitro digestion performance, i.e., an improved activity onPNP-Oleate at pH 5.0 in the presence of 2 mM bile salts, more inparticular an improved bile salt ratio as determined by the method inExample 9.

In a further particular embodiment, the lipase of the invention isselected from amongst lipases having the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N33Q+G91T+G163K+T231R+N233R+D254G;-   N33Q+G91T+G163K+T231R+N233R+D254S; and-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S.

A most preferred lipase comprises the following substitutions,preferably sets of substitutions, as compared to the lipase of SEQ IDNO: 2:

-   N11R+N33Q+G91T+G163K+T231R+N233R+D254S.

In a particular embodiment, the lipase of the invention has an improvedactivity at low pH. In an activity context, low pH means a pH in therange of from 4 to 7, e.g. pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0. Apreferred low pH is pH 6.0. In preferred embodiments, the activity at pH6.0 is determined: i) at 37° C.; ii) with the substrate of trilinolein,preferably in a concentration of 8 mM; iii) with bile salts presentduring the incubation of enzyme and substrate, preferably in aconcentration of 10 mM; iv) using as assay buffer 100 mM imidazole, 100mM acetate, 100 mM malonic acid, pH 6.0; v) with CaCl₂ being presentduring the incubation of enzyme and substrate, preferably in aconcentration of 1 mM; and/or vi) with an amount of purified lipasecorresponding to 0.01 mg EP/mL (EP=enzyme protein, based on A₂₈₀₎). Inadditional preferred embodiments, vii) the enzyme is diluted before theassay (e.g., in order to obtain an appropriate concentration for assaypurposes) in 5 mM NaH₂PO₄ pH 7.0; iix) enzyme and substrate areincubated for 30 minutes; ix) enzyme and substrate are incubated inmicro titer plates (MTP), and preferably shaken with 700 rpm; x) theenzymatic reaction is stopped with a stop solution, preferably (2.2%Triton-X100, 0.22 M Phosphoric acid), more preferably including pepsin(70 mg/l); xi) the free fatty acids generated as a result of the enzymereaction are determined by an enzymatic color test, such as NefaC;and/or xii) the improvement in activity at pH 6.0 is indicated relativeto the activity under the same conditions of a reference lipase such asthe lipase having amino acids 1-269 of SEQ ID NO: 1 or 2, preferably 2,or relative to variant LV2934. For more details regarding the testmethod, please refer to Example 3. Particular examples of lipasevariants of an improved activity at pH 6.0 are (relative to the lipasehaving amino acids 1-269 of SEQ ID NO: 2): LVA049, LVA349, LVA023,LVA099, SEQ ID NO: 1, LVA061, LV2934, LV1330, LVA043, LVA041, LVA012,LV1857, and LV1855 (see Table 1 for their structure). The LVA049,LVA349, LVA023, and LVA099 lipase variants are particularly preferred(improved also as compared to the SEQ ID NO: 1 lipase). The LVA049 andLVA349 lipase variants are even more preferred. A most preferred lipase,from a pH-activity point of view, is the LVA049 lipase variant.

In another particular embodiment, the lipase of the invention has animproved stability at low pH. In a stability context, low pH means a pHin the range of from 2 to 6, e.g., pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, or 6.0. A preferred low pH is 3.0. The stability of a purifiedlipase enzyme is determined by incubating the enzyme at 37° C. at thedesired pH (e.g., 3.0) for 1, 15, 45, and 120 minutes, following whichresidual lipase activity is measured on p-Nitrophenyl caprylate at pH 8and room temperature (RT). In preferred embodiments, i) the buffer usedfor the stability pre-incubation (stability buffer) is 200 mM imidazole,200 mM acetate, 200 mM malonic acid, adjusted to the desired pH (e.g.,3.0); ii) the enzymes are first diluted in 20 mM NaH₂PO₄ pH 7.0, 0.01%Triton-X100 to working solutions of 0.4 or 0.8 mg enzyme protein per ml,preferably based on A₂₈₀, iii) the enzyme concentration during thepre-incubation is 0.05 or 0.1 mg enzyme protein per ml, and for thisdilution the buffer is preferably enzyme dilution buffer: 20 mM acetatepH 6, 0.01% Triton-X100; iv) the pre-incubation is in micro titer plates(MTP) with shaking, preferably with 700 rpm; v) for the subsequentdetermination of residual activity (RA), enzyme-containing aliquotswithdrawn from the pre-incubation step are diluted at least 20 times inthe following residual activity buffer (RA buffer): 200 mM Tris(tris-hydroxymethyl aminomethan, 2-amino-2-hydroxymethyl-1,3-propandiol,pH 8, 0.4% Triton-X100, 1 mM CaCl₂; vi) the residual activity ismeasured on p-Nitrophenyl caprylate at pH 8.0 and RT and is measured byway of kinetics (velocity; rate) at 405 nm for 5 minutes; vii) the %residual activity is calculated as follows: The rate within each pH foreach withdraw (1, 15, 45, 120 minutes; or 1, 60, 120 minutes) issubtracted the rate for no enzyme control, if applicable with bile saltsor pepsin (see below iix) and ix)), and this corrected rate is thendivided by the highest value within each pH and multiplied by 100.Optionally, the enzymes are pre-incubated iix) in the presence of bilesalts, preferably in a concentration of 10 mM, and/or ix) in thepresence of pepsin (70 mg/l). For more details regarding this testmethod, please refer to Example 4.

In further particular embodiments, the lipase of the invention has a %residual activity, determined as described above and in Example 4, of atleast 60, 65, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92 after120 minutes of incubation at pH 3.0 in buffer.

In further particular embodiments, the lipase of the invention has a %residual activity, determined as described above and in Example 4, of atleast 82, 83, 84, 85, 86, 87, 88, or at least 89 after 60 minutes ofincubation at pH 3.0 in buffer.

In still further particular embodiments, the lipase of the invention hasa % residual activity, determined as described above and in Example 4,of at least 35, 40, 45, 50, 55, 60, 65, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, or 94 after 45 minutes of incubation at pH 3.0 in thepresence of pepsin.

In still further particular embodiments, the lipase of the invention hasa % residual activity, determined as described above and in Example 4,of at least 20, 30, 40, 50, 60, 70, 80, 82, 84, 86, 88, or at least 89after 60 minutes of incubation at pH 3.0 in the presence of pepsin.

In still further particular embodiments, the lipase of the invention hasa % residual activity, determined as described above and in Example 4,of at least 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, or at least 71 after120 minutes of incubation at pH 3.0 in the presence of pepsin.

In still further particular embodiments, the lipase of the invention hasa % residual activity, determined as described above and in Example 4,of at least 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, or 50 after 15 minutes of incubation at pH 3.0in the presence of bile salt.

Particular examples of lipase variants of an improved stability at pH3.0 are (relative to the lipase having the sequence of amino acids 1-269of SEQ ID NO: 2): LV2934, LVA043, LVA049, LV1855, LV1865, LV1874,LV1889, LV1857, LVA012, LVA023, LVA041, LVA061, and LVA099. Particularlypreferred lipases having an improved stability at pH 3.0 in the presenceof pepsin are the following: LVA043, LV1855, LV1865, LV1874, LV1889,LV1857, LVA012, and LVA099. Further examples of lipases having animproved stability at pH 3.0 in the presence of pepsin are thefollowing: LVA147, LVA315, LVA317, LVA319, and LVA714. These areimproved as compared to either of the lipases of SEQ ID NO: 2, SEQ IDNO: 1, and LV2934. Another particularly preferred lipase variant whichhas an improved stability at pH 3.0 in the presence of bile salts isLVA349. See Table 1 and Example 4 for the structure of these lipasevariants.

In another particular embodiment, which may be particularly useful forless purified lipase preparations, e.g. for screening purposes, thestability at pH 3.0 is measured as follows: First the enzyme ispre-incubated for 3 hours at pH 3.0 and room temperature in the presenceof 75 ug/mL pepsin, and then the residual lipase activity is measured ina rate assay monitoring activity over time. In preferred embodiments, i)the substrate for the residual activity assay is 4-nitrophenolpalmitate, preferably 1 mM PNP-Palmitate, 1.2% Triton-X100, 4 mM CaCl₂,100 mM TRIS, pH 8.0; ii) for the residual activity assay, OD₄₀₅ readingsare taken from 15 minutes after substrate addition and until 18 hoursafter substrate addition; iii) OD₄₀₅ readings are expressed as mOD(milli OD) per hour; iv) data that falls in the linear range arecollected and the residual lipase activity of each pepsin-treated samplecompared with the residual lipase activity of the correspondinguntreated sample; v) % residual activity (% RA) is calculated bydividing the rate of the treated condition by the rate of the untreatedcondition and multiplying the result by 100. See Example 8 for moredetails. The following variants have an improved stability at pH 3.0 inthe presence of pepsin, as compared to the lipase having the sequence ofamino acids 1-269 of SEQ ID NO: 2: The lipase having amino acids 1-269of SEQ ID NO: 1, LVAR0002b, LVAR0003, LVAR0011a, LVAR0013, LVAR0014,LVAR0015, LVAR0016, LVAR0017, LVAR0045, LVAR0046, LVAR0047, LVAR0048,LVAR0050, LVAR0051, LVAR0052, LVAR0053, LVAR0054, LVAR0055, LVAR0056,LVAR0057, LVAR0058, LVAR0059, LVAR0061, LVAR0062, LVAR0063, LVAR0064,LVAR0065, LVAR0066, LVAR0067, LVAR0068, LVAR0069, LVAR0070, LVAR0071,LVAR0072, LVAR0101, LVAR0102, and LVAR0106. Preferred variants are:LVAR0011a, LVAR0013, LVAR0017, LVAR0046, LVAR0052, LVAR0055, LVAR0061,LVAR0063, LVAR0068, LVAR0070, LVAR0071, LVAR0072, LVAR0014, LVAR0015,LVAR0057, LVAR0101, LVAR0102, and LVAR0106. Particularly preferredlipase variants are: LVAR0014, LVAR0015, LVAR0057, LVAR0101, LVAR0102,and LVAR0106. The structure of these variants is shown in Tables 6 and9.

In another particular embodiment, the lipase of the invention is stablein the presence of pepsin, e.g. in the presence of 70 mg/ml pepsin,preferably for 15, 45, 60, and/or 120 minutes at a desired pH (e.g. pH2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0) and 37° C. For more details,see the above section addressing Example 4.

In a still further particular embodiment, the lipase of the invention isstable in the presence of bile salts, e.g. in the presence of 10 mM bilesalts, preferably for 15, 45, and/or 120 minutes at a desired pH (e.g.pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0) and 37° C. For moredetails, see the above section addressing Example 4.

In a still further particular embodiment, the lipase of the inventionhas an improved phospholipase activity, as compared to a referencelipase such as the lipase having the sequence of amino acids 1-269 ofSEQ ID NO: 2, or the lipase having the sequence of amino acids 1-269 ofSEQ ID NO: 1. The phospholipase activity may be determined as follows:i) the purified enzyme is diluted in enzyme dilution buffer (20 mMNa-Acetate, 0.01% w/w Triton-X100, pH 5.0) to 5 mgEP/ml, e.g. based onA₂₈₀; ii) the activity on1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine is determined,preferably at 40° C. and for 20 minutes; iii) the liberated free fattyacids are determined and quantified by MALDI-TOF MS, preferably aftermixture with 20 mg/mL 2,5-dihydro-benzoic acid in 50% MeOH, 0.1% TFA(matrix); iii) the relative signal intensities (area under each peak) ofthe MS peaks are used for the calculation of the distribution betweenPhospholipase A1 and A2 activity.

Lipases with an improved % undigested phospholipid left after hydrolysisas compared to a reference lipase such as the lipase having amino acids1-269 of SEQ ID NO: 2, or the lipase having amino acids 1-269 of SEQ IDNO: 1, have an improve phospholipase activity.

Particular examples of lipase variants with improved phospholipaseactivity as compared to SEQ ID NO: 2 are the following: LV1889, LVA023,LV1330, LV1855, LV1865, LV1874, LV1889, LVA043, LVA049, LV1857, andLV1232. Preferred lipases are LV1232 and LV1889.

In a still further particular embodiment, the lipase of the inventionhas an improved performance in an in vitro digestion model as comparedto the lipase having amino acids 1-269 of SEQ ID NO: 2, the lipasehaving amino acids 1-269 of SEQ ID NO: 1, and/or as compared to LV2934(the deglycosylated variant N33Q of SEQ ID NO: 1). The in vitro modelmakes use of Diet I, or Diet II, which are described in the Experimentalpart. In brief, 100 ul of diet is mixed with 20 ul pepsin (700 mg/ml)and 30 ul lipase (duplicate of 4 concentrations) in the well of amicrotiter plate, which is incubated for 1 hour at 37° C. with shaking(750 rpm) before adding 25 ul buffer (0.8 M MES(2-[N-morpholino]ethanesulfonic acid), 0.8 M sodium acetate, 0.8 Mimidazole, pH 7.0) and 20 ul bile salts (100 mM) resulting in a pH of5.7 to 6.0. The plate is then incubated 2 hours at 37° C. with agitationbefore stopping the reaction by adding 50 ul 10% Triton-X100 in 1 Mphosphoric acid. After diluting 125-250 times in 1% Triton-X100 theamount of free fatty acids is determined using a colorimetric kit, suchas the NEFA C kit, as described in Example 3.

Examples of lipases of an improved performance in vitro are: LVAR0003,LVAR0045, LVAR0046, LVAR0047, LVAR0050, LVAR0051, LVAR0052, LVAR0053,LVAR0054, LVAR0056, LVAR0057, LVAR0061, LVAR0062, LVAR0063, LVAR0064,LVAR0065, LVAR0067, LVAR0069, and LVAR0072. Other examples are:LVAR0074, LVAR0076, LVAR0077, LVAR0078, LVAR0079, LVAR0080, LVAR0086,LVAR0088, LVAR0091, LVAR0094, LVAR0095, LVAR0096, LVAR0099, LVAR0101,LVAR0102, LVAR0103, LVAR0104, LVAR0106, and LVAR0108. Preferred examplesare: LVAR0003, LVAR0013, LVAR0032, LVAR0050, LVAR0058, and LVAR0069.More preferred are: LVAR0063, LVAR0067, LVAR0069, LVAR0079, LVAR0080,LVAR0094, LVAR0095, LVAR0096, LVAR0099, LVAR0101, LVAR0102, LVAR0103,LVAR0104, LVAR0106, and LVAR0108. Most preferred are LVAR0094, LVAR0099,LVAR0095, and LVAR0106.

In a still further particular embodiment, the lipase of the inventionhas an improved performance in vivo. The in vivo performance may beestimated in a lipase screening test in female Göttingen minipigs(Ellegaard) with induced Pancreatic Exocrine Insufficiency (PEI), asdescribed in Example 10, and/or in a full in vivo digestibility trial asdescribed in Example 11. The performance may be improved relative to thelipase having amino acids 1-269 of SEQ ID NO: 2, the lipase having aminoacids 1-269 of SEQ ID NO: 1, and/or LV2934 being deglycosylated variantN33Q of the lipase having amino acids 1-269 of SEQ ID NO: 1. For moredetails of this test, please see Example 10.

The lipase of the invention preferably comprises at least one of thefollowing substitutions: N26I, D27Q, D27R, D27Y, P29T, A30T, A30V, T321,N33Q, N33T, N33Y, P42L, E43D, E43K, E43M, E43V, A49T, L69I, E87K, E99D,E99K, E99P, E99S, E99T, G163K, S216P, L227G, T231R, N233R, D234K, E239V.

The lipase of the invention preferably comprises at least one of thefollowing substitutions: N26I, D27Q, D27R, D27Y, P29T, A30T, A30V, T32I,N33Q, N33T, N33Y, P42L, E43D, E43K, E43M, E43V, A49T, E56C, E56S, D57A,D57G, D57N, V60L, L69I, E87K, G91A, G91E, G91N, G91R, G91S, G91T, G91V,G91W, L93F, N94K, N94R, N94S, D96E, D96G, D96L, D96N, D96S, D96V, D96W,D96Y, L97M, L97Q, K981, E99D, E99K, E99P, E99S, E99T, D111A, D111S,T114I, L147S, G163K, E210D, S216P, L227G, T231R, N233R, D234K, E239V,Q249R, N251S, D254N, P256T, G263Q, L264A, I265T, G266D, T267A, and/orL269N.

In a particular embodiment, the lipase of the invention is not: (i) thelipase having amino acids 1-269 of SEQ ID NO: 1; (ii) variant N33Q ofthe lipase of (i); (iii) amino acids −5-269 (−5 to +269), −4-269 (−4 to+269), −3-269 (−3 to +269), −2-269 (−2 to +269), −1-269 (−1 to +269),and 2-269 of SEQ ID NO: 1; (iv) variant N33Q of any one of the sequencesof (iii); any one of embodiments (i), (ii), (iii), and/or (iv) with anamino-terminal methionine residue, (v) any one of embodiments (i), (ii),(iii), (iv), and/or (v) with a polyhistidine tract; (vi) any one ofembodiments (i), (ii), (iii), (iv), (v) and/or (vi) with at least oneconservative substitution as defined on p. 5, lines 4-18 of WO2006/136159; (vii) a fragment of any one of the previous embodiments asdefined on p. 6, lines 4-14 of WO 2006/136159; (iix) a specific mixtureof variants as defined on p. 6, line 34 to p. 7, line 11 of WO2006/136159; and/or not (ix) a lipase specifically disclosed forpharmaceutical use in WO 2006/136159.

Particularly preferred lipases of the invention are: LV1232, LV1855,LV1857, LV1865, LV1874, and LV1889.

Other particularly preferred lipases of the invention are the followinglipases, which are variants of a parent lipase, and comprise(T231R+N233R) and in addition at least one of the followingsubstitutions: N26I, D27Q, D27R, D27Y, A30V, T32I, N33Y, P42L, E43K,E43M, E43V, A49T, E56A, E56C, E56K, E56R, E56S, D57A, D57G, D57N, E87K,G91E, G91N, G91R, G91V, G91W, L93F, N94K, N94R, D96G, D96L, D96N, D96S,D96V, D96W, D96Y, L97M, L97Q, K98I, E99K, E99P, E99S, E99T, D111A,D111S, T114I, L147S, G163K, S216P, L227G, D234K, E239V, Q249R, D254N,G2630, L264A, 1265T, G266D, and/or L269N.

Ligase, Protease, Amylase

The following ligases are also included within the scope of the presentinvention: Any one of the lipases claimed and disclosed herein,comprising in addition any one of the following N-terminal extensions:SPIRR, PIRR, IRR, RR, and R, corresponding to amino acids −5 to −1 ofSEQ ID NO: 2, −4 to −1 of SEQ ID NO: 2, −3 to −1 of SEQ ID NO: 2, −2 to−1 of SEQ ID NO: 2, and −1 of SEQ ID NO: 2, respectively. Also anymixture of any of these N-terminal versions is specifically includedherein.

In a particular embodiment, the specific activity of the lipase of theinvention is at least 50% of the specific activity of the lipase havingamino acids 1-269 of SEQ ID NO: 2. In additional particular embodiments,the specific activity of the variant lipase is at least 60, 70, 75, 80,85, 90, or at least 95% of the specific activity of the lipase havingamino acids 1-269 of SEQ ID NO: 2. The specific activity may be measuredusing any of the lipase assays of Example 1 herein, but is preferablymeasured in LU/mg enzyme protein using the LU-assay of Example 1, anddetermining enzyme protein content, e.g. as described in Example 2 (A₂₈₀and GPMAW), or using amino acid analysis. In an amino acid analysis, thepeptide bonds of the lipase sample are subjected to acid hydrolysis,followed by separation and quantification of the released amino acids,e.g. on a Biochrom 20 Plus Amino Acid Analyser, commercially availablefrom Bie & Berntsen A/S, Sandbaekvej 5-7, DK-2610 Roedovre, Denmark,according to the manufacturer's instructions. The amount of eachindividual amino acid is determined by reaction with ninhydrin.

In still further particular embodiments, the lipase of the invention isused in combination with an additional lipase. Examples of additionallipases are mammalian lipases, and microbial lipases. A preferredmammalian lipase is pancreas extract, e.g. from swine or ox, such aspancreatin. The pancreatin may be used in the form of an uncoated (raw)product, or in the form of a formulated product (enteric coated (toprovide resistance against gastric acid), or non-functionally coated(coated, but not to provide resistance against gastric acid)).Pancreatin potentially comprises still further enzymatic activeconstituents like pancreatic protease and/or pancreatic amylase. Themicrobial lipase may be, e.g., based on or derived from a bacterial orfungal lipase. Bacterial lipases can be derived from, e.g., Bacillus orPseudomonas, fungal lipases can be derived from, e.g., strains ofRhizopus, Candida, or Humicola, such as Rhizopus delemar, Rhizopusjavanicus, Rhizopus oryzae, or Humicola lanuginosa, in particular eitherof the products Lipase D2™ or Lipase D Amano 2000™ (lipase, EC 3.1.1.3)which are commercially available from Amano Pharmaceuticals, Japan.

The lipase of the invention may be used in combination with a protease,with or without an amylase as described below. The term “protease” isdefined herein as an enzyme that hydrolyses peptide bonds. It includesany enzyme belonging to the EC 3.4 enzyme group (including each of thethirteen subclasses thereof, these enzymes being in the followingreferred to as “belonging to the EC 3.4.-.- group”).

Examples of proteases are mammalian proteases, and microbial proteases.A preferred mammalian protease is pancreas extract, e.g. from swine orox, such as pancreatin. The pancreatin may be used in the form of anuncoated (raw) product, or in the form of a formulated product (entericcoated, or non-functionally coated). Pancreatin potentially comprisesstill further enzymatic active constituents like pancreatic lipase, BSSL(Bile Salt Stimulated Lipase), and/or pancreatic amylase.

The microbial protease may be, e.g., based on or derived from bacterialor fungal strains. The protease may in particular be derived from astrain of Aspergillus, such as Aspergillus oryzae or Aspergillusmelleus, in particular the product Prozyme 6™ (neutral, alkalineprotease EC 3.4.21.63) which is commercially available from AmanoPharmaceuticals, Japan. Examples of bacterial proteases are proteasesfrom Bacillus and Nocardiopsis, such as the Bacillus licheniformisprotease having the amino acid sequence of amino acids 1-274 of SEQ IDNO: 3, the Nocardiopsis sp. protease having the amino acid sequence ofamino acids 1-188 of SEQ ID NO: 4, or the Nocardiopsis dassonvielleisubsp. dassonvillei protease having the amino acid sequence of aminoacids 1-188 of SEQ ID NO: 5. The protease of amino acids 1-274 of SEQ IDNO: 3 may, e.g., be prepared as described in WO 2006/136160. Theproteases of amino acids 1-188 of SEQ ID NO: 4-5 may, e.g., be preparedas described in WO 2001/58276, or in WO 2004/111224.

In a preferred embodiment, the protease of the invention is at least 70%identical to a protease having, or comprising, either of (i) amino acids1-274 of SEQ ID NO: 3, (ii) amino acids 1-188 of SEQ ID NO: 4, and/or(iii) amino acids 1-188 of SEQ ID NO: 5. In additional preferredembodiments of either of (i), (ii) or (iii), the degrees of identity isat least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99%. In alternative embodiments of either of (i),(ii), or (iii), the degrees of identity is at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, or at least 69%.

The lipase of the invention, with or without a protease as describedabove, may also be used in combination with an amylase.

In the present context, an amylase is an enzyme that catalyzes theendo-hydrolysis of starch and other linear and branched oligo- andpolysaccharides. The amylose part of starch is rich in1,4-alpha-glucosidic linkages, while the amylopectin part is morebranched containing not only 1,4-alpha- but also 1,6-alpha-glucosidiclinkages. In a particular embodiment, the amylase is an enzyme belongingto the EC 3.2.1.1 group.

In particular embodiments, the amylase is a mammalian amylase or amicrobial amylase. An example of a mammalian amylase is pancreasextract, e.g. from swine or ox, such as pancreatin. The pancreatin maybe used in the form of an uncoated (raw) product, or in the form of aformulated product (enteric coated, or non-functionally coated).Pancreatin potentially comprises still further enzymatic activeconstituents like pancreatic protease and/or pancreatic lipase. Themicrobial amylase may be, e.g., based on or derived from bacterial orfungal strains, such as Bacillus, Pseudomonas, Aspergillus, or Rhizopus.

The amylase may in particular be derived from a strain of Aspergillus,such as Aspergillus niger, Aspergillus oryzae or Aspergillus melleus,for example either of the products Amylase A1™ derived from Aspergillusoryzae which is commercially available from Amano Pharmaceuticals,Japan, or Amylase EC™ derived from Aspergillus melleus which iscommercially available from Extract-Chemie, Germany.

Preferred amylases are (i) an amylase comprising amino acids 1-481 ofSEQ ID NO: 6 (such as amino acids 1-481, 1-484, or 1-486 thereof), aminoacids 1-481 of SEQ ID NO: 7, and/or amino acids 1-483 of SEQ ID NO: 8.In a preferred embodiment, the amylase is an amylase having, orcomprising an amino acid sequence being, at least 70% identical toeither of (i) amino acids 1-481 of SEQ ID NO: 6, (ii) amino acids 1-481of SEQ ID NO: 7, and/or (iii) amino acids 1-483 of SEQ ID NO: 8. Theamylases of SEQ ID NOs: 6-8 may, e.g., be prepared as described inco-pending WO 2006/136161. In additional preferred embodiments of eitherof (i), (ii), or (iii), the degrees of identity are at least 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least99%. In alternative embodiments of either of (i), (ii), or (iii), thedegrees of identity are at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or at least 69%.

Generally, the lipase, protease, and amylase enzymes (hereinafter “theenzyme(s),” viz. the enzymes of the invention) may be natural orwild-type enzymes (obtained from animals, in particular mammals, forexample human or swine enzymes; from plants, or from microorganisms),but also any mutants, variants, fragments etc. thereof exhibiting thedesired enzyme activity, as well as synthetic enzymes, such as shuffled,hybrid, or chimeric enzymes, and consensus enzymes.

In a specific embodiment, the enzyme(s) are low-allergenic variants,designed to invoke a reduced immunological response when exposed toanimals, including man. The term immunological response is to beunderstood as any reaction by the immune system of an animal exposed tothe enzyme(s). One type of immunological response is an allergicresponse leading to increased levels of IgE in the exposed animal.Low-allergenic variants may be prepared using techniques known in theart. For example the enzyme(s) may be conjugated with polymer moietiesshielding portions or epitopes of the enzyme(s) involved in animmunological response. Conjugation with polymers may involve in vitrochemical coupling of polymer to the enzyme(s), e.g. as described in WO96/17929, WO 98/30682, WO 98/35026, and/or WO 99/00489. Conjugation mayin addition or alternatively thereto involve in vivo coupling ofpolymers to the enzyme(s). Such conjugation may be achieved by geneticengineering of the nucleotide sequence encoding the enzyme(s), insertingconsensus sequences encoding additional glycosylation sites in theenzyme(s) and expressing the enzyme(s) in a host capable ofglycosylating the enzyme(s), see, e.g., WO 00/26354. Another way ofproviding low-allergenic variants is genetic engineering of thenucleotide sequence encoding the enzyme(s) so as to cause the enzymes toself-oligomerize, effecting that enzyme monomers may shield the epitopesof other enzyme monomers and thereby lowering the antigenicity of theoligomers. Such products and their preparation is described e.g. in WO96/16177. Epitopes involved in an immunological response may beidentified by various methods such as the phage display method describedin WO 00/26230 and WO 01/83559, or the random approach described in EP561907. Once an epitope has been identified, its amino acid sequence maybe altered to produce altered immunological properties of the enzyme(s)by known gene manipulation techniques such as site directed mutagenesis(see, e.g., WO 00/26230, WO 00/26354 and/or WO 00/22103) and/orconjugation of a polymer may be done in sufficient proximity to theepitope for the polymer to shield the epitope.

In particular embodiments, the enzyme(s) are (i) stable at pH 2-8,preferably also at pH 3-7, more preferably at pH 4-6; (ii) active at pH4-9, preferably 4-8; (iii) stable against degradation by pepsin andother digestive proteases (such as pancreas proteases, i.e., mainlytrypsin and chymotrypsin); and/or (iv) stable and/or active in thepresence of bile salts.

The term “in combination with” refers to the combined use according tothe invention of the lipase, protease and/or amylase. The combined usecan be simultaneous, overlapping, or sequential, these three terms beinggenerally interpreted in the light of the prescription made by thephysician.

The term “simultaneous” refers to circumstances under which the enzymesare active at the same time, for example when they are administered atthe same time as one or more separate pharmaceutical products, or ifthey are administered in one and the same pharmaceutical composition.

The term “sequential” refers to such instances where one and/or two ofthe enzymes are acting first, and the second and/or third enzymesubsequently. A sequential action can be obtained by administering theenzymes in question as separate pharmaceutical formulations with desiredintervals, or as one pharmaceutical composition in which the enzymes inquestion are differently formulated (compartmentalized), for examplewith a view to obtaining a different release time, providing an improvedproduct stability, or to optimizing the enzyme dosage.

The term “overlapping” refers to such instances where the enzymeactivity periods are neither completely simultaneous nor completelysequential, viz. there is a certain period in which the enzymes areboth, or all, active.

The term “a”, for example when used in the context of the protease,lipase, and/or amylase of the invention, means at least one. Inparticular embodiments, “a” means “one or more,” or “at least one”,which again means one, two, three, four, five etc.

The activity of the enzyme(s) of the invention can be measured using anysuitable assay. Generally, assay-pH and assay-temperature may be adaptedto the enzyme in question. Examples of assay-pH-values are pH 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperatures are 30, 35,37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95° C. Preferred pH valuesand temperatures are in the physiological range, such as pH values of 4,5, 6, 7, or 8, and temperatures of 30, 35, 37, or 40° C.

Examples of suitable enzyme assays are included in the experimentalpart. Other examples are the FIP or Ph.Eur. assays for protease andamylase activity. These assays are, e.g., described in co-pendingapplications WO 2006/136160 and WO 2006/136161, respectively.

Medicament

In the present context, the term “medicament” means a compound, ormixture of compounds, that treats, prevents and/or alleviates thesymptoms of disease, preferably treats and/or alleviates the symptoms ofdisease. The medicament may be prescribed by a physician, or it may bean over-the-counter product.

Pharmaceutical Compositions

Isolation, purification, and concentration of the enzyme(s) of theinvention may be carried out by conventional means. For example, theymay be recovered from a fermentation broth by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation, and further purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulphate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

For example, a variant of the lipase of SEQ ID NO: 2 such as the lipaseof SEQ ID NO: 1 may, e.g., be prepared on the basis of U.S. Pat. No.5,869,438 (in which SEQ ID NO: 1 is a DNA sequence encoding the lipaseof SEQ ID NO: 2 herein), viz. by recombinant expression in a suitablehost cell of a DNA sequence which is a modification of SEQ ID NO: 1 ofthe US patent, the modification reflecting the amino acid differencesbetween SEQ ID NO: 1 and 2 herein. Such modifications can be made bysite-directed mutagenesis, as is known in the art.

In a particular embodiment, concentrated solid or liquid preparations ofeach of the enzyme(s) are prepared separately. These concentrates mayalso, at least in part, be separately formulated, as explained in moredetail below.

In a further particular embodiment, the enzyme(s) are incorporated inthe pharmaceutical compositions of the invention in the form of solidconcentrates. The enzyme(s) can be brought into the solid state byvarious methods as is known in the art. For example, the solid state canbe either crystalline, where the enzyme molecules are arranged in ahighly ordered form, or a precipitate, where the enzyme molecules arearranged in a less ordered, or disordered, form.

Crystallization may, for example, be carried out at a pH close to the pIof the enzyme(s) and at low conductivity, for example 10 mS/cm or less,as described in EP 691982. In a particular embodiment, the lipase foruse according to the invention is a crystalline lipase, which can beprepared as described in Example 1 of EP 600868 B1. The lipase crystalsmay furthermore be cross-linked as described in WO 2006/044529.

Various precipitation methods are known in the art, includingprecipitation with salts, such as ammonium sulphate, and/or sodiumsulphate; with organic solvents, such as ethanol, and/or isopropanol; orwith polymers, such as PEG (Poly Ethylene Glycol). In the alternative,the enzyme(s) can be precipitated from a solution by removing thesolvent (typically water) by various methods known in the art, e.g.lyophilization, evaporation (for example at reduced pressure), and/orspray drying.

In a further particular embodiment, the solid concentrate of theenzyme(s) has a content of active enzyme protein of at least 50% (w/w)by reference to the total protein content of the solid concentrate. Instill further particular embodiments, the content of active enzymeprotein, relative to the total protein content of the solid concentrateis at least 55, 60, 65, 70, 75, 80, 85, 90, or at least 95% (w/w). Theprotein content can be measured as is known in the art, for example bydensitometer scanning of coomassie-stained SDS-PAGE gels, e.g. using aGS-800 calibrated densitometer from BIO-RAD; by using a commercial kit,such as Protein Assay ESL, order no. 1767003, which is commerciallyavailable from Roche; or on the basis of the method described in Example8 of WO 01/58276.

Preferably, the enzyme protein (e.g., lipase enzyme protein) constitutesat least 50%, more preferably at least 55, 60, 65, 70, 75, 80, 85, 90,92, 94, 95, 96, or at least 97% of the protein spectrum of the solidenzyme concentrate for use according to the invention, as measured bydensitometer scanning of a coomassie-stained SDS-PAGE gel. Such enzymesmay be designated “isolated”, “purified”, or “purified and isolated”enzymes or polypeptides. For the lipase expressed in Aspergillus andcomprising a mixture of various N-terminal forms as explained in Example5 of WO 2006/136159, the relevant band on an SDS-PAGE gel is locatedcorresponding to a molecular weight of 34-40 kDa. For a non-glycosylatedvariant such as N33Q of SEQ ID NO: 1 (LV2934), the relevant band islocated at around 30 kDa.

A pharmaceutical composition of the invention comprises the enzyme(s),preferably in the form of concentrated enzyme preparations, morepreferably solid concentrates, together with at least onepharmaceutically acceptable auxiliary, or subsidiary, material such as(i) at least one carrier and/or excipient; or (ii) at least one carrier,excipient, diluent, and/or adjuvant. Non-limiting examples of, optional,other ingredients, all pharmaceutically acceptable, are disintegrators,lubricants, buffering agents, moisturizing agents, preservatives,flavouring agents, solvents, solubilizing agents, suspending agents,emulsifiers, stabilizers, propellants, and vehicles.

Generally, depending i.a. on the medical indication in question, thecomposition of the invention may be designed for all manners ofadministration known in the art, preferably including enteraladministration (through the alimentary canal). Thus, the composition maybe in solid, semi-solid, liquid, or gaseous form, such as tablets,capsules, powders, granules, microspheres, ointments, creams, foams,solutions, suppositories, injections, inhalants, gels, lotions, andaerosols. The medical practitioner will know to select the most suitableroute of administration and of course avoid potentially dangerous orotherwise disadvantageous administration routes.

The following methods and auxiliary materials are therefore also merelyexemplary and are in no way limiting.

For solid oral preparations, the enzyme(s) can be used alone or incombination with appropriate additives to make pellets, micropellets,tablets, microtablets, powders, granules or capsules, for example, withconventional carriers, such as lactose, mannitol, corn starch, or potatostarch; with excipients or binders, such as crystalline, ormicrocrystalline, cellulose, cellulose derivatives, acacia, corn starch,or gelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as carnauba wax,white wax, shellac, waterless colloid silica, polyethylene glycol (PEGs,also known under the term macrogol) from 1500 to 20000, in particularPEG4000, PEG6000, PEG 8000, povidone, talc, monolein, or magnesiumstearate; and if desired, with diluents, adjuvants, buffering agents,moistening agents, preservatives such as methylparahydroxybenzoate(E218), colouring agents such as titanium dioxide (E171), and flavouringagents such as saccharose, saccharin, orange oil, lemon oil, andvanillin. Oral preparations are examples of preferred preparations fortreatment of the medical indication of PEI.

The enzyme(s) can also, quite generally, be formulated into liquid oralpreparations, by dissolving, suspending, or emulsifying them in anaqueous solvent such as water, or in non-aqueous solvents such asvegetable or other similar oils, synthetic aliphatic acid glycerides,esters of higher aliphatic acids, propylene glycol, polyethylene glycolsuch as PEG4000, or lower alcohols such as linear or ramified C1-C4alcohols, for example 2-propanol; and if desired, with conventionalsubsidiary materials or additives such as solubilizers, adjuvants,diluents, isotonic agents, suspending agents, emulsifying agents,stabilizers, and preservatives.

Furthermore, the enzyme(s) can generally be made into suppositories forrectal administration by mixing with a variety of bases such asemulsifying bases or water-soluble bases. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

The use of liposomes as a delivery vehicle is another method of possiblegeneral interest. The liposomes fuse with the cells of the target siteand deliver the contents of the lumen intracellularly. The liposomes aremaintained in contact with the cells for sufficient time for fusion,using various means to maintain contact, such as isolation, bindingagents, and the like. In one aspect of the invention, liposomes aredesigned to be aerosolized for pulmonary administration. Liposomes maybe prepared with purified proteins or peptides that mediate fusion ofmembranes, such as Sendai virus or influenza virus, etc. The lipids maybe any useful combination of known liposome forming lipids, includingcationic or zwitterionic lipids, such as phosphatidylcholine. Theremaining lipid will normally be neutral or acidic lipids, such ascholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.For preparing the liposomes, the procedure described by Kato et al.,1991, J. Biol. Chem. 266:3361 may be used.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, powders, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, capsule, tablet orsuppository, contains a predetermined amount of the enzyme(s).Similarly, unit dosage forms for injection or intravenous administrationmay comprise the enzyme(s) in a composition as a solution in sterilewater, normal saline, or another pharmaceutically acceptable carrier.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of enzyme(s) inan amount sufficient to produce the desired effect.

In a particular embodiment, the pharmaceutical composition of theinvention is for enteral, preferably oral, administration.

In further particular embodiments, the oral composition is (i) a liquidcomposition containing crystals of the enzyme(s); (ii) a liquidsuspension of sediments of (highly) purified enzyme(s); (iii) a gelcontaining the enzyme(s) in solid or solubilized form; (iv) a liquidsuspension of immobilized enzyme(s) or of enzymes adsorbed to particlesand the like; or (v) a solid composition in the form ofenzyme(s)-containing powder, pellets, granules, or microspheres, ifdesired in the form of tablets, capsules, or the like, that areoptionally coated, for example with an acid-stable coating.

In another particular embodiment of the composition, the enzyme(s) arecompartmentalized, viz. separated from each other, for example by meansof separate coatings.

In a still further particular embodiment of the composition, theprotease is separated from other enzyme components of the composition,such as the lipase, and/or the amylase.

The dosage of the enzyme(s) will vary widely, depending on the specificenzyme(s) to be administered, the frequency of administration, themanner of administration, the severity of the symptoms, and thesusceptibility of the subject to side effects, and the like. Some of thespecific enzymes may be more potent than others.

Examples of solid oral preparations of the enzyme(s) of the inventioncomprise: (i) a lipase of the invention; (ii) a protease having at least70% identity to a protease selected from the group consisting of a) aprotease having amino acids 1-274 of SEQ ID NO: 3, b) a protease havingamino acids 1-188 of SEQ ID NO: 4, and c) a protease having amino acids1-188 of SEQ ID NO: 5; and/or (iii) an amylase having at least 70%identity to an amylase selected from the group consisting of a) anamylase having amino acids 1-481 of SEQ ID NO: 6, b) an amylase havingamino acids 1-481 of SEQ ID NO: 7, and c) an amylase having amino acids1-483 of SEQ ID NO: 8; wherein preferably the anticipated daily clinicaldosages of the enzymes of (i), (ii), and (iii) are as follows (all in mgenzyme protein per kg of bodyweight (bw)): For the lipase of (i):0.01-1000, 0.05-500, 0.1-250, or 0.5-100 mg/kg bw; for the amylase of(ii): 0.001-250, 0.005-100, 0.01-50, or 0.05-10 mg/kg bw; for theprotease of (iii): 0.005-500, 0.01-250, 0.05-100, or 0.1-50 mg/kg bw.

A preferred example of solid oral preparations of the enzyme(s) of theinvention comprise: (i) a lipase of the invention, and (ii) an amylasecomprising amino acids 1-481 of SEQ ID NO: 6, and/or (iii) a proteasecomprising, preferably having, amino acids 1-274 of SEQ ID NO: 3.

Examples of anticipated daily clinical dosages of the enzymes of (i),(ii), and (iii) are as follows (all in mg enzyme protein per kg ofbodyweight (bw)): For the lipase of (i): 0.1-250, 0.5-100, or 1-50 mg/kgbw; for the amylase of (ii): 0.01-50, 0.05-10, or 0.1-5 mg/kg bw; forthe protease of (iii): 0.05-100, 0.1-50, or 0.5-25 mg/kg bw.

The amide (peptide) bonds, as well as the amino and carboxy termini, maybe modified for greater stability on oral administration. For example,the carboxy terminus may be amidated.

Particular embodiments of pharmaceutical compositions of the invention,suitable for the treatment of digestive disorders, PEI, pancreatitis,cystic fibrosis, diabetes type I, and/or diabetes type II, may beprepared by incorporating the enzyme(s) of the invention into pellets.The pellets may generally comprise from 10-90% (w/w, relative to the dryweight of the resulting pellets) of a physiologically acceptable organicpolymer, from 10-90% (w/w, relative to the dry weight of the resultingpellets) of cellulose or a cellulose derivative, and from 80-20% (w/w,relative to the dry weight of the resulting pellets) of the enzyme(s),the total amount of organic polymer, cellulose or cellulose derivativeand enzyme(s) making up to 100% in each case.

The physiologically acceptable organic polymer can be selected from thegroup consisting of polyethylene glycol 1500, polyethylene glycol 2000,polyethylene glycol 3000, polyethylene glycol 4000, polyethylene glycol6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethyleneglycol 20000, hydroxypropyl methylcellulose, polyoxyethylene, copolymersof polyoxyethylene-polyoxypropylene and mixtures of said organicpolymers. Polyethylene glycol 4000 is preferred as physiologicallyacceptable organic polymer.

The cellulose or a cellulose derivative can e.g. be selected fromcellulose, cellulose acetate, cellulose fatty acid ester, cellulosenitrates, cellulose ether, carboxymethyl cellulose, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose,methyl ethylcellulose and methylhydroxypropyl cellulose. Cellulose, inparticular microcrystalline cellulose is preferred as cellulose orcellulose derivative.

The resulting pellets may be coated with a suitable enteric coating,other non functional coating or be used directly without such coating.Further, the resulting pellets may be filled in capsules like hardgelatin capsules or gelatin free capsules of a suitable size for therapyof a disorder or disease as described in more detail above. In anembodiment of the invention, pellets produced from different enzymetypes, in particular from lipase, protease and/or amylase may be filledinto said capsules. While filling the capsules with the different enzymetypes, the dosing of the single enzyme types (viz. lipase, protease oramylase) may be adapted to specific needs of a certain indication groupor a certain patient subgroup by adding a specified amount of any oflipase, protease and/or amylase to the capsules, i.e., capsules may beproduced which vary in their specific ratios of lipase:protease:amylase.

Preferred pharmaceutical compositions of the lipase of the invention aredescribed in WO 2005/092370, in particular formulations comprising thepreferred exhibients mentioned therein. In a particularly preferredembodiment, the pharmaceutical composition comprises a macrogolglyceridemixture of mono-, di- and tri-acylglycerides and polyethylene glycol(PEG) mono- and di-esters of aliphatic C6-C22 carboxylic acids, and alsopossibly small proportions of glycerol and free polyethylene glycol.

The polyethylene glycol (PEG) contained in the macrogolglyceridemixtures is preferably PEG which has on average 6 to at most 40 ethyleneoxide units per molecule or a molecular weight of between 200 and 2000.

One further aspect of the invention provides for the pharmaceuticalcomposition of the enzyme(s) of the invention to comprise a systemconsisting of surfactant, co-surfactant and lipophilic phase, the systemhaving an LVB value (Hydrophilic-Lipophilic Balance) greater than orequal to 10 and a melting point greater than or equal to 30° C. In apreferred embodiment, the system has an LVB value of 10 to 16,preferably of 12 to 15, and has a melting point of between 30 and 600°C., preferably between 40 and 500° C. In particular, the systemcharacterised by LVB value and melting point is a mixture of mono-, di-and triacylgylcerides and mono- and diesters of polyethylene glycol(PEG) with aliphatic carboxylic acids with 8 to 20, preferably 8 to 18,carbon atoms, whereby the polyethylene glycol preferably has about 6 toabout 32 ethylene oxide units per molecule, and the system optionallycontains free glycerin and/or free polyethylene glycol. The LVB value ofsuch a system is preferably regulated by the chain length of the PEG.The melting point of such a system is regulated by the chain length ofthe fatty acids, the chain length of the PEG and the degree ofsaturation of the fatty-acid chains, and hence the starting oil for thepreparation of the macrogolglyceride mixture.

“Aliphatic C8-C18 carboxylic acids” designates mixtures in whichcaprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid(C14), palmitic acid (C16) and stearic acid (C18) are contained in asignificant and variable proportion, if these acids are saturated, andthe corresponding unsaturated C8-C18 carboxylic acids. The proportionsof these fatty acids may vary according to the starting oils.

Such a mixture of mono-, di- and triacylgylcerides and mono- anddiesters of polyethylene glycol (PEG) with aliphatic carboxylic acidswith 8 to 18 carbon atoms can for example be obtained by a reactionbetween a polyethylene glycol with a molecular weight of between 200 and1500 and a starting oil, the starting oil consisting of a triglyceridemixture with fatty acids which are selected from the group containingcaprylic acid, capric acid, lauric acid, myristic acid, palmitic acid,stearic acid, oleic acid and linolenic acid, individually or as amixture. Optionally, the product of such a reaction may also containsmall proportions of glycerin and free polyethylene glycol.

Such mixtures are commercially available for example under the tradename Gelucire®. One advantageous embodiment of the invention providesthat, of the products known under the trade name Gelucire®, inparticular “Gelucire® 50/13” and/or “Gelucire® 44/14” represent suitablemixtures for use in the pharmaceutical preparations according to theinvention.

Gelucire® 50/13 is a mixture with mono-, di- and triacylglycerides andmono- and diesters of polyethylene glycol, with palmitic acid (C16) andstearic acid (C18) at 40% to 50% and 48% to 58%, respectively making upthe major proportion of bound fatty acids. The proportion of caprylicacid (C8) and capric acid (C10) is less than 3% in each case, and theproportion of lauric acid (C12) and myristic acid (C14) in each case isless than 5%.

Gelucire® 44/14 is a mixture with mono-, di- and triacylgylcerides andmono- and diesters of polyethylene glycol, the respective proportions ofpalmitic acid (C16) being 4 to 25%, stearic acid (C18) 5 to 35%,caprylic acid (C8) less than 15%, capric acid (C10) less than 12%,lauric acid (C12) 30 to 50% and myristic acid (C14) 5 to 25%. Gelucire®44/14 can for example be prepared by an alcoholysis/esterificationreaction using palm kernel oil and polyethylene glycol 1500.

A preferred embodiment of the present invention provides for apharmaceutical composition of the enzyme(s) of the invention whichcomprises a system containing a mixture of mono-, di- andtriacyl-glycerides and polyethylene glycol mono- and diesters ofaliphatic C8-C18 carboxylic acids and also possibly small proportions ofglycerin and free polyethylene glycol, the system having a melting pointbetween 40° C. and 55° C. and an LVB value in the range between 12 and15. More preferred, the system has a melting point between 44° C. and50° C. and an LVB value in the range from 13-14. Alternatively, thesystem has a melting point around 44° C. and an LVB value of 14, or thesystem has a melting point around 50° C. and an LVB value of 13.

Methods of Treatment

The lipase for use according to the invention, optionally in combinationwith a protease, and/or an amylase (the enzyme(s) of the invention), isuseful in the therapeutic, and/or prophylactic, treatment of variousdiseases or disorders in animals. The term “animal” includes allanimals, and in particular human beings. Examples of animals arenon-ruminants, and ruminants, such as sheep, goat, and cattle, e.g. beefcattle, and cow. In a particular embodiment, the animal is anon-ruminant animal. Non-ruminant animals include mono-gastric animals,e.g. horse, pig (including, but not limited to, piglets, growing pigs,and sows); poultry such as turkey, duck and chicken (including but notlimited to broiler chicks, layers); young calves; pets such as cat, anddog; and fish (including but not limited to salmon, trout, tilapia,catfish and carps; and crustaceans (including but not limited to shrimpsand prawns). In a particular embodiment the animal is a mammal, more inparticular a human being.

For example, the enzyme(s) are useful in the treatment of digestivedisorders like maldigestion or dyspepsia that are often caused by adeficient production and/or secretion into the gastrointestinal tract ofdigestive enzymes normally secreted from the stomach, and the pancreas.

Further, the enzyme(s) are particularly useful in the treatment of PEI.PEI can be verified using, i.a., the Borgström test (JOP. J Pancreas(Online), 2002; 3(5):116-125), and it may be caused by diseases andconditions such as pancreatic cancer, pancreatic and/or gastric surgery,e.g. total or partial resection of the pancreas, gastrectomy, postgastrointestinal bypass surgery (e.g. Billroth II gastroenterostomy);chronic pancreatitis; Shwachman Diamond Syndrome; ductal obstruction ofthe pancreas or common bile duct (e.g. from neoplasm); and/or cysticfibrosis (an inherited disease in which a thick mucus blocks the ductsof the pancreas). The enzyme(s) may also be useful in the treatment ofacute pancreatitis.

The effect of the enzyme(s) on digestive disorders can be measured asgenerally described in EP 0600868, in which Example 2 describes an invitro digestibility test for measuring lipase stability under gastricconditions, and Example 3 an in vitro digestibility test for lipaseactivity in the presence of bile salts. Corresponding tests can be setup for the protease and amylase. Also WO 02/060474 discloses suitabletests, for example (1) an in vitro test for measuring lipid digestion ina swine test feed, and (2) an in vivo trial with pancreas insufficientswine in which the digestibility of fat, protein and starch is measured.

As another example, the enzyme(s) are useful in the treatment ofDiabetes mellitus type I, and/or type II, in particular for adjuvanttreatment in a diabetes therapy of digestive disorders usuallyaccompanying this disease, with a view to diminishing latecomplications.

The effect on Diabetes mellitus of the enzyme(s) may be determined byone or more of the methods described in WO 00/54799, for example bycontrolling the level of glycosylated haemoglobin, the blood glucoselevel, hypoglycemic attacks, the status of fat-soluble vitamins likevitamins A, D and E, the required daily dosage of insulin, thebody-weight index, and hyper glycaemic periods.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Particular Embodiments

The invention also relates to a lipase, preferably for use as amedicament, which lipase, as compared to the sequence of amino acids1-269 of SEQ ID NO:2, comprises the substitutions of any one of claims1-2 and 5-9, such as substitutions N33Q, T231R, and N233R, as well as atleast one additional substitution selected from the following:

-   E1*,D,N; Q4H,P,R; D5E; N8L,Q; Q9H; F10L; N11C,D,H,L,P,Q,R,S; G23E;    N26A,H,I; D27I,N,Q,R,S,V; P29T; A30T,V; T37K,M;    G38A,D,F,H,I,K,L,M,N,P,Q,S,T,W,Y; N39H,S; E43K; K46M; A49T; L52I, R;    E56K,Q,R,S; D57G,N; V60E,S; G61R; V63R; A68V; L69I; N71I, S; N73Q,Y;    I76T; R84E; I86F,L; E87A,H,K,R; I90L,V;    G91A,C,E,F,K,L,M,N,S,T,V,W,Y; L93*,F; N94*,K,Q,R,S; F95*;    D96*,E,G,N,R,S,W,Y; L97M,Q; K98I,T; E99D; N101Q; D102E,G,Y; R108M;    G109A; D111A,E,N,S; G112A; T114I; S115L;    W117C,D,E,F,G,H,I,K,L,P,S,T,V,Y; D122E,N; Q126L; V128A; D130H;    H135D; P136H; Y138F; V141E,L; A150V; V154F,I,L; A155V; G156R;    G161A,E; N162G,S,T; G163A,C,D,E,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; D167E;    V168M; V176A,D,F,G,H,I,K,M,N,Q,T,W; G177A; R179T; L185M; G190C,D;    N200Q,S; R205I; L206F; E210D,R,V,Y; S216P; E219D; G225P; T226N;    L227F,G; P229R; E239D; G240L; D242E; T244S; G246A; Q249R; N251Q,S;    D254A,G,I,K,L,M,N,R,Q,S,Y; I255A,F; P256A,F,G,H,I,L,M,N,Q,S,T,V,W,Y;    and L269F,H; said lipase furthermore:

(a) having at least 50% identity to the sequence of amino acids 1 to 269of SEQ ID NO: 2;

(b) being encoded by a polynucleotide that hybridizes under very low(preferably low, medium, medium-high, high, or most preferably veryhigh) stringency conditions with (i) the coding sequence of SEQ ID NO: 1of U.S. Pat. No. 5,869,438 which is hereby incorporated by reference (aDNA sequence encoding the lipase of SEQ ID NO: 2 herein), or (ii) afull-length complementary strand of (i); and/or

(c) being a variant comprising in addition a substitution, deletion,and/or insertion of one or more (e.g., several) amino acids of themature polypeptide of SEQ ID NO: 1, preferably of a conservative nature.

Very low to very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200microg/ml sheared and denatured salmon sperm DNA, and either 25%formamide for very low and low stringencies, 35% formamide for mediumand medium-high stringencies, or 50% formamide for high and very highstringencies, following standard Southern blotting procedures for 12 to24 hours optimally. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very lowstringency), more preferably at 50° C. (low stringency), more preferablyat 55° C. (medium stringency), more preferably at 60° C. (medium-highstringency), even more preferably at 65° C. (high stringency), and mostpreferably at 70° C. (very high stringency).

Amino acid changes of a conservative nature do not significantly affectthe folding and/or activity of the protein, and include small deletions,typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

The invention furthermore relates to a variant of a parent lipase,preferably for use as a medicament, which variant comprises analteration at one or more positions, said positions corresponding to oneor more positions in the parent enzyme wherein:

(a) the alteration(s) are independently

-   -   (i) an insertion of an amino acid immediately downstream of the        position,    -   (ii) a deletion of the amino acid which occupies the position,        and/or    -   (iii) a substitution of the amino acid which occupies the        position;

(b) the alterations are selected from the alterations of any one ofclaims 1-2 and 5-9;

(c) the variant has lipase activity; and

(d) each position corresponds to a position of the amino acid sequenceof the enzyme having the amino acid sequence of SEQ ID NO: 2.

In a particular embodiment, the variant, and/or the parent has at least50% identity to the sequence of amino acids 1 to 269 of SEQ ID NO: 2.

The total number of alterations in the variant preferably is twenty-two,twenty-one, twenty, nineteen, eighteen, seventeen, or sixteen. Morepreferably the total number of alterations is fifteen, even morepreferably fourteen, even more preferably thirteen, even more preferablytwelve, even more preferably eleven, even more preferably ten, even morepreferably nine, even more preferably eight, even more preferably seven,even more preferably six, even more preferably five, even morepreferably four, even more preferably three, even more preferably two,and most preferably one.

A variant may be produced by shuffling one or more polynucleotidesencoding one or more homologous parent lipases. The term “shuffling”means recombination of nucleotide sequence(s) between two or morehomologous nucleotide sequences resulting in recombined nucleotidesequences (i.e., nucleotide sequences having been subjected to ashuffling cycle) having a number of nucleotides exchanged, in comparisonto the starting nucleotide sequence.

The following variants of the lipase of SEQ ID NO: 2 are examples oflipases of claim 6 and 9:

LVA012 = D27R + N33Q + G91A + D96E + L97Q + D111A + LVA013 T231R +N233R + P256T; LVA023 N33Q + E210D + T231R + N233R; LVA041 N33Q +D111A + T231R + N233R; LVA043 N33Q + G91T + T231R + N233R; LVA055 N33Q +E219D + T231R + N233R; LVA060 N33Q + W117L + T231R + N233R; LVA061D27Q + N33Q + T231R + N233R; LVA063 N33Q + G91T + T231R + N233R; LVA089D27S + N33Q + G91A + D96E + L97Q + D111A + S216P + T231R + N233R +P256T; LVA094 D27R + N33Q + G91N + N94R + D111A + T231R + N233R + P256T;LVA099 D27R + N33Q + G91T + N94S + D111A + S216P + L227G + T231R +N233R + P256T; LVA103 Q4R + N33Q + T231R + N233R; LVA113 N33Q + T231R +N233R + Q249R; LVA120 N33Q + D96W + T231R + N233R; LVA129 D27V + N33Q +V60S + D96W + T231R + N233R + Q249R; LVA130 D27V + N33Q + V60S + T231R +N233R + Q249R; LVA139 Q9H + N33Q + D102E + T231R + N233R; LVA140 N33Q +D111E + T231R + N233R; LVA143 N33Q + D122E + T231R + N233R; LVA147D27R + N33Q + G91N + N94R + D111A + S216P + L227G + T231R + N233R +P256T; LVA162 N33Q + D167E + T231R + N233R; LVA179 N33Q + G91N + T231R +N233R; LVA180 N33Q + T231R + N233R + P256T; LVA182 D27R + N33Q + G91A +L93* + N94* + F95* + D96* + D111A + T231R + N233R + P256T; LVA185 N11R +N33Q + T231R + N233R; LVA198 N33Q + N39H + T231R + N233R; LVA202 N33Q +P229R + T231R + N233R; LVA206 D27R + N33Q + G91N + N94R + D111A +G163K + S216P + L227G + T231R + N233R + P256T; LVA208 N33Q + G91T +G163K + T231R + N233R; LVA210 D27R + N33Q + G91A + D96E + L97Q + D111A +S216P + L227G + T231R + N233R +P256T; LVA211 D27R + N33Q + G91A + D96E +L97Q + D111A + S216P + T231R + N233R + P256T; LVA214 N33Q + E87A +T231R + N233R; LVA216 N33Q + E56Q + T231R + N233R; LVA217 N33Q + E210V +T231R + N233R; LVA218 N33Q + E56K + T231R + N233R; LVA220 N33Q + T231R +N233R + D254G; LVA221 N33Q + D96S + T231R + N233R; LVA222 N33Q + D122N +T231R + N233R; LVA228 N26A + N33Q + T231R + N233R; LVA229 N33Q + N162T +T231R + N233R; LVA230 N33Q + A150V + N162G + T231R + N233R; LVA231N33Q + I90L + G163L + T231R + N233R; LVA234 N33Q + T231R + N233R +G240L; LVA238 D27R + N33Q + G91A + D96E + D111A + T231R + N233R +D254G + P256T; LVA241 D27R + N33Q + G91A + N94S + D111A + T231R +N233R + P256T; LVA243 N33Q + N200S + T231R + N233R; LVA245 N33Q + N39S +T231R + N233R; LVA247 N33Q + E210R + T231R + N233R; LVA248 N33Q + N39H +T231R + N233R + D254R; LVA249 N33Q + T231R + N233R + D254R; LVA250N33Q + N94R + T231R + N233R; LVA252 N33Q + D96R + T231R + N233R; LVA254D27N + N33Q + T231R + N233R; LVA256 D27N + N33Q + E56R + T231R + N233R;LVA257 N33Q + L227F + T231R + N233R; LVA272 N33Q + N73Y + G225P +T231R + N233R; LVA273 N33Q + G225P + T231R + N233R; LVA275 N33Q +T231R + N233R + D254S; LVA277 N33Q + D96G + T231R + N233R; LVA279 N33Q +D96N + T231R + N233R + D254S; LVA280 N33Q + T231R + N233R + D254G;LVA281 N33Q + D130H + T231R + N233R; LVA284 N33Q + E87A + T231R + N233R;LVA287 N33Q + T231R + N233R + E239D; LVA307 N33Q + D111A + T231R +N233R + D254G; LVA308 N33Q + E210V + T231R + N233R + D254S; LVA310N11R + N33Q + E210V + T231R + N233R + D254S; LVA315 N33Q + G91T +G163K + T231R + N233R + D254G; LVA317 N33Q + G91T + G163K + T231R +N233R + D254S; LVA319 N11R + N33Q + G91T + G163K + T231R + N233R +D254S; LVA325 Q4R + D27R + N33Q + G91T + N94S + D111A + S216P + L227G +T231R + N233R + P256T; LVA327 N33Q + G91T + N94S + D111A + V176I +T231R + N233R; LVA330 Q4R + D27R + N33Q + G91T + N94S + D111A + E210D +S216P + L227G + T231R + N233R + P256T; LVA331 Q4R + D27Q + N33Q + G91T +N94S + D111A + S216P + L227G + T231R + N233R + P256T; LVA333 N33Q +G91T + N94S + D111A + T231R + N233R + P256T; LVA334 N33Q + G177A +T231R + N233R; LVA338 N33Q + T231R + N233R + G246A; LVA341 D27N + N33Q +G91T + G163K + T231R + N233R + D254S; LVA345 D27Q + N33Q + G91T +G163K + E219D + T231R + N233R; LVA347 N33Q + G91T + E219D + T231R +N233R; LVA349 K98I + T231R + N233R + N251S; LVA353 N33Q + G163R +T231R + N233R; LVA355 N33Q + G163N + T231R + N233R; LVA357 N33Q +G163C + T231R + N233R; LVA359 N33Q + G163Q + T231R + N233R; LVA360 =N33Q + G163E + T231R + N233R; LVA415 LVA362 N33Q + G163H + T231R +N233R; LVA364 N33Q + G163I + T231R + N233R; LVA368 N33Q + G163P +T231R + N233R; LVA370 N33Q + G163D + T231R + N233R; LVA371 N33Q + G91K +T231R + N233R; LVA373 N33Q + G91M + T231R + N233R; LVA375 N33Q + G91F +T231R + N233R; LVA379 N33Q + G91S + T231R + N233R; LVA381 N33Q + G91W +T231R + N233R; LVA383 N33Q + G91Y + T231R + N233R; LVA387 N33Q + G163T +T231R + N233R; LVA389 N33Q + G163W + T231R + N233R; LVA391 N33Q +G163Y + T231R + N233R; LVA393 N33Q + G163V + T231R + N233R; LVA399N33Q + G91C + T231R + N233R; LVA411 N33Q + G91Y + Q126L + T231R + N233R;LVA412 N33Q + G91M + G161E + T231R + N233R; LVA413 = N33Q + V128A +T231R + N233R; LVA414 LVA416 N33Q + G163V + L185M + T231R + N233R;LVA417 N33Q + G38A + T231R + N233R; LVA420 N33Q + G163A + T231R + N233R;LVA421 N33Q + G91T + N94S + D111A + T231R + N233R; LVA437 N33Q + G38A +G163A + T231R + N233R; LVA438 N33Q + G163M + T231R + N233R; LVA440N33Q + G91V + T231R + N233R; LVA442 N33Q + D111A + T231R + N233R +Q249R; LVA444 N33Q + D111A + T231R + N233R + D254S; LVA449 D27R + N33Q +G91A + D96E + L97Q + D111A + T231R + N233R + D254S + P256T; LVA450D27R + N33Q + G91A + D96E + L97Q + D111A + T231R + N233R + D254G +P256T; LVA451 N33Q + G91T + N94R + T231R + N233R + D254S; LVA453 N33Q +G91T + N94R + D111A + W117L + T231R + N233R; LVA454 N33Q + W117L +T231R + N233R + D254S; LVA456 N33Q + T231R + N233R + P256T; LVA458N33Q + T231R + N233R + D242E; LVA460 N33Q + E87R + T231R + N233R; LVA461N33Q + E56R + T231R + N233R; LVA463 N33Q + N162G + T231R + N233R; LVA464N33Q + G91L + T231R + N233R; LVA468 N33Q + E87H + T231R + N233R; LVA470N33Q + D96N + T231R + N233R + Q249R; LVA471 N33Q + G91T + N94R + T231R +N233R + D254S; LVA472 N33Q + L227F + T231R + N233R + D254S; LVA473D27R + N33Q + G91T + D96E + L97Q + D111A + T231R + N233R + D254S +P256T; LVA474 N33Q + G163A + T231R + N233R; LVA480 D27R + N33Q + G91T +D96E + D111A + T231R + N233R + D254S + P256T; LVA482 N33Q + G91T +N94R + T231R + N233R; LVA483 N33Q + T231R + N233R + D254A; LVA484 N33Q +T231R + N233R + D254N; LVA486 N33Q + T231R + N233R + D254Q; LVA488N33Q + T231R + N233R + D254I; LVA490 N33Q + T231R + N233R + D254L;LVA492 N33Q + T231R + N233R + D254K; LVA494 N33Q + T231R + N233R +D254M; LVA503 N33Q + S216P + L227G + T231R + N233R + Q249R; LVA505D27V + N33Q + V60S + G91T + D96W + T231R + N233R + Q249R; LVA506 N33Q +D96N + L227G + T231R + N233R + Q249R; LVA507 D27R + N33Q + L227G +T231R + N233R; LVA509 D27R + N33Q + L227G + T231R + N233R + Q249R;LVA512 N33Q + E219D + L227G + T231R + N233R + Q249R; LVA513 D27Q +N33Q + L227G + T231R + N233R + Q249R; LVA516 N33Q + W117L + L227G +T231R + N233R + Q249R; LVA518 D5E + N33Q + W117L + L227G + T231R +N233R + Q249R; LVA519 D27Q + N33Q + E219D + L227G + T231R + N233R +Q249R; LVA520 N33Q + D96E + E219D + L227G + T231R + N233R + Q249R;LVA523 D27R + N33Q + E56K + G91N + N94R + D111A + S216P + L227G +T231R + N233R + P256T; LVA526 D27R + N33Q + E56Q + D57N + G91N + N94R +D111A + S216P + L227G + T231R + N233R + P256T; LVA527 D27R + N33Q +E56Q + D57N + G91N + N94R + D111S + S216P + L227G + T231R + N233R +D254S + P256T; LVA530 D27R + N33Q + E56S + G91N + N94R + D111A + S216P +L227G + T231R + N233R + P256T; LVA532 D27R + N33Q + G91N + N94R +D111A + S216P + L227G + T231R + N233R + D254S + P256T; LVA535 D27R +N33Q + G91N + N94R + D111A + S216P + L227G + T231R + N233R + D254S +P256T; LVA540 D27R + N33Q + G91N + N94R + D111S + A155V + S216P +L227G + T231R + N233R + D254S + P256T; LVA542 D27R + N33Q + G91N +N94R + D111S + S216P + L227G + T231R + N233R + D254S + P256T; LVA547N33Q + D111A + T231R + N233R + D254S; LVA548 N33Q + D111A + W117L +T231R + N233R + D254S; LVA553 N33Q + T231R + N233R + P256A; LVA555N33Q + T231R + N233R + P256N; LVA561 N33Q + T231R + N233R + P256G;LVA562 N33Q + T231R + N233R + P256H; LVA564 N33Q + T231R + N233R +P256L; LVA565 N33Q + T231R + N233R + P256M; LVA566 N33Q + T231R +N233R + P256S; LVA567 N33Q + T231R + N233R + P256W; LVA569 N33Q +T231R + N233R + P256Y; LVA576 N33Q + T231R + N233R + P256F; LVA578N33Q + T231R + N233R + P256V; LVA580 N33Q + G91M + G163W + T231R +N233R; LVA581 N33Q + G91M + G163T + T231R + N233R; LVA582 N33Q + G91M +G163D + T231R + N233R; LVA583 N33Q + G91K + G163W + T231R + N233R;LVA586 N33Q + G91T + G163W + T231R + N233R; LVA602 N33Q + V176N +T231R + N233R; LVA604 N33Q + V176D + T231R + N233R; LVA614 N33Q +W117F + T231R + N233R; LVA620 N33Q + G91T + N94S + D111A + V176I +T231R + N233R + D254S; LVA622 N33Q + V176I + T231R + N233R; LVA623N33Q + D111N + T231R + N233R; LVA627 N33Q + D111N + G225P + T231R +N233R; LVA629 N33Q + D111N + S216P + T231R + N233R; LVA631 D27R + N33Q +G91T + N94R + D111A + S216P + L227G + T231R + N233R; LVA632 N33Q +G91M + G163P + T231R + N233R; LVA634 N33Q + G91T + G163A + T231R +N233R; LVA639 N33Q + W117D + T231R + N233R; LVA640 N33Q + W117H +T231R + N233R; LVA649 = N33Q + W117C + T231R + N233R; LVA650 LVA651N33Q + W117K + T231R + N233R; LVA653 N33Q + W117V + T231R + N233R;LVA656 N11S + N33Q + T231R + N233R; LVA658 N33Q + W117E + V176K +T231R + N233R; LVA659 N33Q + W117G + T231R + N233R; LVA664 N33Q +W117P + T231R + N233R; LVA665 N33Q + W117S + T231R + N233R; LVA666N33Q + W117T + T231R + N233R; LVA667 N33Q + W117I + T231R + N233R;LVA670 D27R + N33Q + L227G + T231R + N233R + Q249R + D254S; LVA672N33Q + S115L + T231R + N233R; LVA675 N33Q + G38A + G91T + G163K +T231R + N233R + D254S; LVA696 N33Q + V176M + T231R + N233R; LVA697N33Q + V176H + T231R + N233R; LVA700 N33Q + V176A + T231R + N233R;LVA702 D27V + N33Q + L227F + T231R + N233R + Q249R; LVA705 N33Q +W117Y + T231R + N233R; LVA707 N33Q + W117Y + V176D + T231R + N233R;LVA713 D27V + N33Q + G91A + N94R + D111A + G163K + L227F + T231R +N233R + Q249R; LVA714 = D27V + N33Q + G91A + N94R + D111A + G163K +LVAR714 L227F + T231R + N233R + Q249R + D254S; LVA715 D27R + N33Q +P136H + L227G + T231R + N233R + Q249R + D254S; LVA718 N11R + N33Q +T231R + N233R + T244S; LVA721 N33Q + G91T + D96N + D111A + V176I +T231R + N233R + D254S; LVA722 N33Q + G91T + N94S + D111A + V176I +T231R + N233R + D254S; LVA723 N33Q + G161A + T231R + N233R; LVA731N33Q + G38I + G177A + T231R + N233R; LVA732 N33Q + N101Q + T231R +N233R; LVA733 N33Q + N94Q + T231R + N233R; LVA734 N33Q + G161A + T231R +N233R; LVA736 N11Q + N33Q + T231R + N233R; LVA738 N8Q + N33Q + T231R +N233R; LVA740 N33Q + T231R + N233R + N251Q; LVA743 N33Q + N200Q +T231R + N233R; LVA744 N33Q + G177A + T231R + N233R; LVA746 N33Q + N73Q +T231R + N233R; LVA749 N33Q + I86L + T231R + N233R; LVA753 N33Q + K98I +G163K + T231R + N233R; LVA754 D27R + N33Q + G91T + D96E + D111A +G163K + T231R + N233R + D254S + P256T; LVA755 D27R + N33Q + G91T +D96E + D111A + G163A + T231R + N233R + D254S + P256T; LVA770 D27R +N33Q + S216P + L227G + T231R + N233R + Q249R; LVA771 N33Q + K98I +G163K + N200Q + T231R + N233R + N251S; LVA772 N33Q + G38S + G163K +T231R + N233R; LVA773 D27R + N33Q + G38A + G91T + D96E + D111A + T231R +N233R + D254S + P256T; LVA774 N33Q G38Y T231R N233R; LVA777 D27R +N33Q + G91T + N94R + D111A + S216P + L227G + T231R + N233R + P256T;LVA778 D27R + N33Q + G91T + N94R + D111A + S216P + L227G + T231R +N233R + P256T; LVA782 N33Q + G38N + N73Q + T231R + N233R; LVA783 N33Q +G38D + R84E + T231R + N233R; LVA784 N33Q + G38Q + T231R + N233R; LVA786N33Q + G38I + T231R + N233R; LVA788 N33Q + G38K + T231R + N233R; LVA792N33Q + G38F + T231R + N233R; LVA799 N33Q + G38H + N200Q + T231R +N233R + N251S; LVA800 N33Q + G38L + T231R + N233R; LVA801 N33Q + G38M +T231R + N233R; LVA803 N33Q + G38F + T231R + N233R; LVA804 N33Q + G38P +T231R + N233R; LVA805 N33Q + G38T + T231R + N233R; LVA806 N11R + N33Q +G91T + W117I + G163K + T231R + N233R + D254S; LVA808 D27R + N33Q +G38A + G91T + D96E + D111A + G163K + T231R + N233R + D254S + P256T;LVA809 N11R + N33Q + G91T + W117I + G163K + T231R + N233R + D254S;LVA811 D27R + N33Q + G38A + G91T + D96E + D111A + G163A + T231R +N233R + D254S + P256T; LVA813 D27R + N33Q + V176Q + L227G + T231R +N233R + Q249R + D254S; LVA814 N33Q + W117I + V176Q + T231R + N233R +P256A; LVA816 N33Q + G38A + G163A + T231R + N233R + P256A; LVA817 N33Q +W117I + V176Q + T231R + N233R; LVA818 N33Q + G177A + T231R + N233R +G246A; LVA819 E1N + N33Q + T231R + N233R; LVA821 N33Q G38H T231R N233R;LVA829 N33Q + G91A + N94K + D111A + G163K + L227F + T231R + N233R +Q249R + D254S; LVA830 N11R + N33Q + G91T + G163K + V176Q + T231R +N233R + D254S; LVA831 N33Q + K98I + T231R + N233R; LVA834 D27R + N33Q +W117I + V176Q + L227G + T231R + N233R + Q249R + D254S; LVA835 N11R +N33Q + G38A + G91T + G163K + T231R + N233R + D254S; LVA839 N33Q +G163W + T231R + N233R; LVA841 N33Q + G38A + G163A + T231R + N233R;LVA842 D27R + N33Q + G91T + D96E + L97Q + D111A + T231R + N233R +D254S + P256T; LVA844 N33Q + T231R + N233R + D254Q; LVA846 N11R + N33Q +G91T + S115L + G163K + T231R + N233R + D254S; LVA847 N11R + N33Q +G91T + G163K + V176W + T231R + N233R + D254S; LVA848 = N33Q + G163D +T231R + N233R; LVA849 LVA850 N33Q + G163P + T231R + N233R; LVA853 E1D +N33Q + G91T + N94R + D111A + W117L + T231R + N233R + D254S; LVA857N33Q + G91T + N94R + D111A + W117L + V176W + T231R + N233R; LVA860 Q4P +D27R + N33Q + G91N + N94R + D111A + L206F + S216P + L227G + T231R +N233R + P256T; LVA862 D27R + N33Q + T37K + N71I + G91N + N94R + K98I +D111A + S216P + L227G + T231R + N233R + P256T; LVA863 D27R + N33Q +E43K + K46M + I90V + G91N + N94R + D111A + T114I + S216P + L227G +T231R + N233R + P256T; LVA865 N33Q + W117S + T231R + N233R; LVA866N33Q + G61R + V63R + G156R + V176W + T231R + N233R + P256I; LVA869N33Q + D96N + G156R + V176W + T231R + N233R; LVA871 N33Q + G156R +V176W + T231R + N233R + Q249R; LVA873 N33Q + G91T + N94S + D111A +G163T + V176W + T231R + N233R; LVA875 N33Q + G91T + N94S + D111A +S115L + G163T + V176I + T231R + N233R; LVA877 N11R + D27R + N33Q +E56Q + D57N + G91N + N94R + D111S + G163T + S216P + L227G + T231R +N233R + D254S + P256T; LVA878 D27R + N33Q + E56Q + D57N + G91N + N94R +D111S + G163T + S216P + L227G + T231R + N233R + D254S + P256T; LVA880N11R + D27R + N33Q + E56Q + D57N + G91N + N94R + D111S + S216P + L227G +T231R + N233R + D254S + P256T; LVA882 D27R + N33Q + E56Q + D57N + G91N +N94R + D111S + S216P + L227G + T231R + N233R + D242E + D254S + P256T;LVA883 D27R + N33Q + G38A + E56Q + D57N + G91N + N94R + D111S + S216P +L227G + T231R + N233R + D254S + P256T; LVA888 Q4R + D27Q + N33Q + G91T +N94S + E99D + D111A + E210D + S216P + L227G + T231R + N233R + P256L;LVA890 N33Q + G38A + G91T + G163A + T231R + N233R + D254S; LVA892 N33Q +G38A + G163A + T231R + N233R + D254I; LVA896 N11R + N33Q + I90L +G163L + T231R + N233R; LVA897 N11R + N33Q + I90L + G163L + T231R +N233R + D254S; LVA899 N11R + N33Q + E56Q + G91T + G163K + V176Q +T231R + N233R + D254S; LVA904 N11R + D27R + N33Q + G91T + D96E + D111A +G163K + T231R + N233R + D254S + P256T; LVA906 N11R + N33Q + G38A +G91T + G112A + G163A + T231R + N233R + D254S; LVA907 N11R + N33Q +G91T + G163K + E210D + T231R + N233R + D254S; LVA913 N11R + N33Q +G91T + G163K + T231R + N233R + D254I; LVA915 N11R + N33Q + G91T +G163K + V176T + T231R + N233R + D254S; LVA917 N11R + N33Q + G91T +G163P + T231R + N233R + D254S; LVA919 N11R + N33Q + G91M + G163T +T231R + N233R + D254S; LVA921 N11R + N33Q + G38A + G91T + G163K +V176D + T231R + N233R + D254S; LVA925 N33Q + E56Q + G156R + V176W +T231R + N233R; LVA927 E1D + N33Q + G38A + G91T + N94R + D111A + W117L +V176W + T231R + N233R; LVA928 N33Q + G163K + G177A + T231R + N233R +G246A; LVA929 N11R + N33Q + E56Q + G91T + G163K + T231R + N233R + D254S;LVA930 N11R + N33Q + I90L + G163K + T231R + N233R + D254S; LVA933 D27R +N33Q + E56Q + D57N + G91N + N94R + D111S + S216P + L227G + T231R +N233R + Q249R + D254S + P256T; LVA934 D27R + N33Q + E56Q + D57N + G91N +N94R + D111S + S216P + E219D + L227G + T231R + N233R + D254S + P256T;LVA941 N11R + N33Q + I90L + G91T + N94S + D96E + G163K + T231R + N233R +D254S; LVA942 N11R + N33Q + G91T + G163K + V176I + T231R + N233R +D254S; LVA943 N11R + N33Q + G91T + G163K + V176Q + T231R + N233R +D254S; LVA944 N11R + N33Q + G91T + G163A + V176T + T231R + N233R +D254S; LVA945 N11R + N33Q + G91T + G163L + V176I + T231R + N233R +D254S; LVA946 N11R + N33Q + G91T + G163L + V176T + T231R + N233R +D254S; LVA947 N11R + N33Q + G91T + G163L + T231R + N233R + D254S; LVA948N11R + N33Q + G91T + G163P + T231R + N233R + D254S; LVA949 N11R + N33Q +G91T + G163P + V176I + T231R + N233R + D254S; LVA950 N11R + N33Q +G91T + G163L + T231R + N233R + D254S + P256N; LVA952 D27R + N33Q +E56Q + D57N + G91N + N94R + D111S + G163T + S216P + L227G + T231R +N233R + Q249R + D254S + P256T; LVA953 Q4R + D27Q + N33Q + G91T + N94S +E99D + D111A + G163A + E210V + S216P + L227G + T231R + N233R + P256L;LVA954 Q4R + D27Q + N33Q + G91T + N94S + E99D + D111A + V176I + E210V +S216P + L227G + T231R + N233R + P256L; LVA959 N33Q + E210Y + T231R +N233R + D254Y + I255F; LVA961 N33Q + L93F + D102Y + T231R + N233R;LVA962 D27R + N33Q + L227G + T231R + N233R + Q249R + D254S; LVA964N11S + N33Q + T231R + N233R; LVA966 N11R + N33Q + T231R + N233R; LVA968N33Q + G38A + G91T + G163K + T231R + N233R + D254S; LVA969 N33Q +W117Y + V176T + T231R + N233R; LVA970 N8L + N11R + N33Q + G91T + G163K +T231R + N233R + D254S; LVA972 E1N + N33Q + G38A + G91T + G163P + V176F +T231R + N233R; LVA973 N11R + N33Q + G38A + G91T + G163P + V176G +T231R + N233R + D254S; LVA976 N11R + N33Q + G91T + G163K + T231R +N233R + D254A + P256F; LVA977 N11R + N33Q + G91T + G163K + T231R +N233R + P256F; LVA978 N11R + N33Q + G91T + G163K + T231R + N233R +D254S + P256F; LVA979 N11R + N33Q + G38A + G91T + G156R + G163K +V176T + T231R + N233R + D254S; LVA980 N33Q + G91K + D96S + G163T +T231R + N233R + Q249R; LVA981 N11R + N33Q + G91T + G163N + T231R +N233R + D254S; LVA983 N11R + N33Q + G91T + G163T + T231R + N233R +D254S; LVA984 N11R + N33Q + G91T + G163W + T231R + N233R + D254S; LVA985N11R + N33Q + G91K + G163K + T231R + N233R + D254S; LVA987 N11R + G23E +N33Q + G91T + G163K + T231R + N233R + D254S; LVA988 N11R + N33Q + G91T +V141E + G163K + T231R + N233R + D254S; LVA989 N11R + N33Q + L52R +G91T + G163K + T231R + N233R + D254S; LVA990 N11R + N33Q + G91T +V141L + G163K + T231R + N233R + D254S; LVA991 N11R + N33Q + T37K +G91T + G163K + T231R + N233R + D254S; LVA993 N11R + N33Q + A68V + G91T +G163K + T231R + N233R + D254S; LVA994 N11R + N33Q + G91T + G163A +V176I + T231R + N233R + D254S; LVA995 N11R + N33Q + T37M + G91T +G163P + V176T + T231R + N233R + D254S; LVA997 N11R + N33Q + G91T +G163L + T231R + N233R + D254S; LVA998 N11R + N33Q + G91T + G163K +T231R + N233R + D254S + P256I; LVA999 N33Q + G38S + G156R + G163K +V176W + T231R + N233R; LVA1000 N11R + D27R + N33Q + E56Q + D57N + G91N +N94R + D111S + G163K + S216P + L227G + T231R + N233R + D254S + P256T;LVA1002 N11R + N33Q + G38A + G91T + G163P + V176G + T231R + N233R +D254S; LVA1003 N11R + N33Q + G38A + G91T + G163Q + V176G + T231R +N233R + D254S; LVA1004 N11R + N33Q + G38A + G91T + G163T + V176G +T231R + N233R + D254S; LVA1005 N11R + N33Q + G38A + G91T + N94R +G163P + V176G + T231R + N233R + D254S; LVA1006 E1* + N11R + N33Q +G38A + G91N + N94R + G163P + V176G + T231R + N233R + D254S; LVA1007E1N + N11R + N33Q + G38A + G91T + G163P + V176F + T231R + N233R; LVA1008E1N + F10L + N11R + N33Q + G38A + G91T + G163P + V176F + T231R + N233R;LVA1009 E1N + N33Q + G38A + G91T + G163P + V176F + T231R + N233R +D254S; LVA1010 E1N + N33Q + G38A + G91T + D111A + G163P + V176F +T231R + N233R; LVA1011 E1N + N33Q + G38A + G91T + G163P + V176F +L227F + T231R + N233R; LVA1012 E1N + N11R + N33Q + G38A + G91T + D111A +G163P + V176F + T231R + N233R; LVA1013 E1N + N33Q + G38A + G91T +G163P + V176F + L227F + T231R + N233R + D254S; LVA1014 E1N + N33Q +G38A + G91T + G163P + V176F + T231R + N233R + D254S + I255A + P256Q;LVA1015 E1N + N11R + N33Q + G38A + G91T + D111A + G163P + V176F +T231R + N233R + D254S; LVA1017 N33Q + G156R + V176W + T231R + N233R +P256I; LVA1018 N33Q + G91T + N94S + D111A + G156R + G163T + V176W +T231R + N233R; LVA1019 N33Q + G91T + N94S + D111A + G156R + G163T +V176I + T231R + N233R; LVA1021 N11R + N33Q + G38A + G91T + D102G +S115L + G163K + T231R + N233R + D254S + P256T; LVA1023 N11R + N33Q +G38A + G91T + S115L + G163K + T231R + N233R + D254S + P256T; LVA1027E1N + N11R + N33Q + G91T + G163A + T231R + N233R + G246A + D254S;LVA1028 N11R + D27R + N33Q + D57G + G91T + D96E + D111A + G163K +T231R + N233R + D254S + P256T; LVA1029 N33Q + D96N + G156R + V176W +T231R + N233R + Q249R; LVA1031 N33Q + I86F + L93F + D102Y + E210Y +L227F + T231R + N233R + D254Y + I255F + L269F; LVA1032 N33Q + I86F +L93F + D102Y + E210Y + L227F + T231R + N233R + D254Y + I255F; LVA1033N11C + N33Q + G91T + G163K + T231R + N233R + D254S; LVA1034 N11L +N33Q + G91T + G163K + T231R + N233R + D254S; LVA1035 N11H + N33Q +G91T + G163K + T231R + N233R + D254S; LVA1036 N11D + N33Q + G91T +G163K + T231R + N233R + D254S; LVA1037 N11R + N33Q + G91T + D96W +G163K + T231R + N233R + D254S; LVA1038 D27R + N33Q + G91T + D96E +L97Q + D111A + G163K + T231R + N233R + D254S + P256T; LVA1040 N11P +N33Q + G91T + G163K + T231R + N233R + D254S; LVA1041 Q4R + D27N + N33Q +G38A + G91T + N94S + E99D + D111A + V176I + E210V + S216P + L227G +T231R + N233R + P256L; LVA1044 N11R + N33Q + E56Q + G163K + T231R +N233R + D254S; LVA1045 N11R + N33Q + G91T + G163A + T231R + N233R +D254S; LVA1046 N11R + N33Q + G91T + G163P + T231R + N233R + D254S;LVA1048 N11R + N33Q + G91T + G163K + L227G + P229R + T231R + N233R +D254S; LVAR0074 N33Q + E87K + T231R + N233R; LVAR0076 N33Q + N94K +T231R + N233R; LVAR0077 N33Q + D96Y + T231R + N233R; LVAR0079 N33Q +K98I + T231R + N233R; LVAR0080 A30V + N33Q + K98I + T231R + N233R;LVAR0086 N33Q + E87K + D96E + T231R + N233R; LVAR0088 N26I + N33Q +T231R + N233R; LVAR0091 A30T + N33Q + T231R + N233R; LVAR0094 N33Q +G91V + T231R + N233R; LVAR0095 N33Q + G91A + T231R + N233R; LVAR0096N33Q + G91V + L97M + T231R + N233R; LVAR0099 N33Q + K98I + T231R +N233R; LVAR0101 N33Q + L69I + G91E + T231R + N233R; LVAR0102 P29T +N33Q + T231R + N233R; LVAR0103 N33Q + G91V + T231R + N233R; LVAR0104N33Q + K98I + T231R + N233R; LVAR0106 N33Q + G91E + T231R + N233R;LVAR0108 N33Q + N94K + T231R + N233R; LVAR204 D27R + N33Q + G91N +N94R + K98I + D111A + N162S + S216P + L227G + T231R + N233R + P256T;LVAR205 D27R + N33Q + T37K + N71I + G91N + N94R + K98I + D111A + S216P +L227G + T231R + N233R + P256T; LVAR207 D27R + N33Q + N39S + G91N +N94R + D111A + S216P + L227G + T231R + N233R + P256T; LVAR208 D27R +N33Q + I76T + G91N + N94R + R108M + D111A + S216P + L227G + T231R +N233R + P256T; LVAR209 D27R + N33Q + L52I + V60E + G91N + N94R + D111A +T114I + V168M + E210D + S216P + L227G + T231R + N233R + P256T; LVAR214Q4P + D27R + N33Q + G91N + N94R + D111A + R205I + L206F + S216P +L227G + T231R + N233R + P256T; LVAR215 Q4H + D27R + N33Q + G91N + N94R +D111A + V154L + S216P + L227G + T231R + N233R + P256T; LVAR216 D27R +N33Q + G91N + N94R + D111A + V154I + S216P + L227G + T231R + N233R +P256T; LVAR218 D27R + N33Q + N71S + G91N + N94R + D111A + H135D +S216P + L227G + T231R + N233R + P256T; LVAR219 D27R + N33Q + G91N +N94R + K98I + D111A + S216P + L227G + T231R + N233R + P256T; LVAR220D27R + N33Q + G91N + N94R + L97M + D111A + S216P + T226N + L227G +T231R + N233R + P256T + L269H; LVAR223 D27R + N33Q + G91N + N94R +D111A + T114I + R179T + S216P + L227G + T231R + N233R + P256T; LVAR225D27R + N33Q + G91N + N94R + D111A + S216P + L227G + T231R + N233RLVAR226 G23E + D27R + N33Q + L52R + G91N + N94R + D111A + T114I +V141E + S216P + L227G + T231R + N233R + P256T; LVAR230 D27R + N33Q +E43K + K46M + I90V + G91N + N94R + D111A + T114I + S216P + L227G +T231R + N233R + P256T; LVAR231 D27R + A30V + N33Q + G91N + N94R +G109A + D111A + G190D + S216P + L227G + T231R + N233R + P256T; LVAR234D27R + N33Q + A49T + G91N + N94R + D111A + Y138F + G163R + S216P +L227G + T231R + N233R + P256T; LVAR235 N26H + D27R + N33Q + G91N +N94R + D111A + V154F + G190C + S216P + L227G + T231R + N233R + P256T;LVAR277 N33Q + G91T + D96E + K98T + T114I + G163S + E210V + T231R +N233R + D254K + P256A; LVAR280 N33Q + G91T + D96E + K98T + T114I +T231R + N233R + G163S; LVAR281 N33Q + G91T + D96E + K98T + T114I +G163K + E210D + T231R + N233R; LVAR282 N33Q + G91T + T114I + G163K +E210D + T231R + N233R + D254G + P256A; LVAR283 D27R + N33Q + G91T +T114I + G163W + E210D + T231R + N233R; LVAR284 D27N + N33Q + G91T +T114I + G163S + E210D + T231R + N233R + P256T; LVAR285 N33Q + G91T +T114I + G163K + E210D + T231R + N233R; LVAR286 N33Q + G38W + G91T +T114I + G163K + E210V + T231R + N233R; LVAR287 N33Q + G38W + G91T +T114I + G163K + E210D + T231R + N233R + P256T; LVAR288 D27I + N33Q +G91T + D96E + K98T + T114I + G163K + E210D + T231R + N233R + P256T;LVAR290 N33Q + G91T + T114I + E210V + T231R + N233R + D254K + P256A;LVAR828 = N33Q + G91A + N94K + D111A + G163K + L227F + LVA828 T231R +N233R + Q249R; LVAR861 G23E + D27R + N33Q + L52R + G91N + N94R + D111A +T114I + V141E + S216P + L227G + T231R + N233R + P256T; LVAR863 D27R +N33Q + E43K + K46M + I90V + G91N + N94R + D111A + T114I + S216P +L227G + T231R + N233R + P256T; LVAR955 N33Q + G91T + K98I + T114I +G163K + T231R + N233R + D254S; LVAR956 N33Q + G91T + K98I + G163K +T231R + N233R + D254S + P256L; LVA957 N33Q + G91T + T114I + G163K +T231R + N233R + D254S + P256L; LVAR1042 G23E + D27R + N33Q + L52R +G91N + N94R + D111A + T114I + V141E + S216P + L227G + T231R + N233R +P256T; and LVAR1043 D27R + N33Q + E43K + K46M + I90V + G91N + N94R +D111A + T114I + S216P + L227G + T231R + N233R + P256T.

The following are additional particular embodiments of the invention(the lipase of SEQ ID NO: 1 is variant (T231R+N233R) of the lipase ofamino acids 1-269 of SEQ ID NO: 2):

-   1. A lipase for use as a medicament, wherein the lipase is a variant    of a parent lipase, which variant (a) has at least 50% identity to    amino acids 1 to 269 of SEQ ID NO: 2; and (b) has lipase activity;    and (c) comprises at least one substitution selected from the    following substitutions: N26I, D27Q, D27R, D27Y, P29T, A30T, A30V,    T32I, N33Q, N33T, N33Y, P42L, E43D, E43K, E43M, E43V, A49T, E56A,    E56C, E56K, E56R, E56S, D57A, D57A, D57G, D57N, V60L, L69I, E87K,    G91A, G91E, G91N, G91R, G91S, G91T, G91V, G91W, L93F, N94K, N94R,    N94S, D96E, D96G, D96L, D96N, D96S, D96V, D96W, D96Y, L97M, L97Q,    K98I, E99D, E99K, E99P, E99S, E99T, D111A, D111S, T114I, L147S,    G163K, E210D, S216P, L227G, T231R, N233R, D234K, E239V, Q249R,    N251S, D254N, P256T, G263Q, L264A, 1265T, G266D, T267A, and L269N,    wherein each position corresponds to a position of amino acids 1 to    269 of SEQ ID NO: 2; and (d) with the proviso that the variant is    not (i) the lipase having amino acids 1-269 of SEQ ID NO: 1, and    not (ii) variant N33Q of the lipase of (i).-   2. A lipase for use as a medicament, wherein the lipase is a variant    of a parent lipase, which variant (a) has at least 50% identity to    amino acids 1 to 269 of SEQ ID NO: 2; and (b) has lipase activity;    and (c) comprises substitutions T231R and N233R and furthermore at    least one substitution selected from the following substitutions:    N26I, D27Q, D27R, D27Y, P29T, A30T, A30V, T321, N33Q, N33T, N33Y,    P42L, E43D, E43K, E43M, E43V, A49T, E56A, E56C, E56K, E56R, E56S,    D57A, D57G, D57N, V60L, L69I, E87K, G91A, G91E, G91N, G91R, G91S,    G91T, G91V, G91W, L93F, N94K, N94R, N94S, D96E, D96G, D96L, D96N,    D96S, D96V, D96W, D96Y, L97M, L97Q, K98I, E99D, E99K, E99P, E99S,    E99T, D111A, D111S, T114I, L147S, G163K, E210D, S216P, L227G, D234K,    E239V, Q249R, N251S, D254N, P256T, G263Q, L264A, I265T, G266D,    T267A, and L269N, wherein each position corresponds to a position of    amino acids 1 to 269 of SEQ ID NO: 2; and (d) with the proviso that    the lipase is not variant N33Q of amino acids 1-269 of SEQ ID NO: 1.-   3. A lipase for use as a medicament, wherein the lipase is a variant    of a parent lipase, which variant (a) has at least 50% identity to    amino acids 1 to 269 of SEQ ID NO: 2; and (b) has lipase activity;    and (c) comprises at least one substitution in at least one of    positions 30, 42, 114, and/or 163, wherein each position corresponds    to a position of amino acids 1 to 269 of SEQ ID NO: 2.-   4. A lipase for use as a medicament, wherein the lipase is a variant    of a parent lipase, which variant (a) has at least 50% identity to    amino acids 1 to 269 of SEQ ID NO: 2; and (b) has lipase activity;    and (c) comprises at least one substitution selected from the    following substitutions: A30T, A30V, P42L, T114I, and G163K, wherein    each position corresponds to a position of amino acids 1 to 269 of    SEQ ID NO: 2.-   5. A lipase for use as a medicament, wherein the lipase is a variant    of a parent lipase, which variant (a) has at least 50% identity to    amino acids 1 to 269 of SEQ ID NO: 2; and (b) has lipase activity;    and (c) is selected from the following variants:

LVA012: D27R + N33Q + G91A + D96E + L97Q + D111A + T231R + N233R +P256T, LVA023: N33Q + E210D + T231R + N233R, LVA041: N33Q + D111A +T231R + N233R, LVA043: N33Q + G91T + T231R + N233R, LVA049: N33Q +G163K + T231R + N233R, LVA061: D27Q + N33Q + T231R + N233R, LVA099:D27R + N33Q + G91T + N94S + D111A + S216P + L227G + T231R + N233R +P256T, LVA349: K98I + T231R + N233R + N251S, LV1232: G91A + D96W +E99K + G263Q + L264A + I265T + G266D + T267A + L269N, LV1330: N33Q +D96S + T231R + N233R + Q249R, LV1855: D27R + G91A + D111A + S216P +L227G + P256T, LV1857: D27R + G91N + N94R + D111A + S216P + L227G +P256T, LV1865: D27R + G91T + N94S + D111A + S216P + L227G + P256T,LV1874: D27R + G91S + D111A + S216P + L227G + P256T, LV1889: D27R +G91T + D96N + D111A + S216P + L227G + P256T, LVAR0002b T32I + G91V +T231R + N233R, LVAR0003: K98I + T231R + N233R, LVAR0011a G91A + T231R +N233R, LVAR0013: G91V + T231R + N233R, LVAR0014 N33Y + G91W + N94K +T231R + N233R, LVAR0015 P42L + D57N + G91E + T231R + N233R, LVAR0016K98I + T231R + N233R, LVAR0017 V60L + G91V + T231R + N233R, LVAR0032:D57G + L93F + T231R + N233R, LVAR0045: A49T + E56R + E87K + E99S +T231R + N233R, LVAR0046: E99T + T114I + D254N + T231R + N233R, LVAR0047:D27Y + E87K + D96L + E99P + T231R + N233R, LVAR0048 E43K + E56S + E87K +T231R + N233R, LVAR0050: E56S ++ E87K + D96L + E99D + T231R + N233R,LVAR0051: E56A + D57A + T114I + T231R + N233R, LVAR0052: G91E + T231R +N233R, LVAR0053: E56K + D96G + D111A + T231R + N233R, LVAR0054: E87K +D111S + T231R + N233R, LVAR0055 E43V + G91R + T231R + N233R, LVAR0056:E56S + E87K + T231R + N233R, LVAR0057: E87K + G91E + T231R + N233R,LVAR0058: D27Y + E87K + T231R + N233R, LVAR0059 E43M + E87K + D96L +E99P + T231R + N233R, LVAR0061: E56K + E87K + D111A + T231R + N233R,LVAR0062: E87K + E99P + T231R + N233R, LVAR0063: E87K + D96L + E99P +T231R + N233R, LVAR0064: E56C + E87K + T231R + N233R, LVAR0065: E56R +E87K + D96L + T231R + N233R, LVAR0066 E43D + E56A + D57A + E87K +D111A + T231R + N233R, LVAR0067: E56K + E87K + D96L + E99P + T231R +N233R, LVAR0068 E87K + L147S + T231R + N233R, LVAR0069: D27Y + E87K +D96L + E99P + T231R + N233R, LVAR0070 E43D + E87K + D96L + E99P +E239V + T231R + N233R, LVAR0071 E43K + E56A + E87K + D234K + T231R +N233R, LVAR0072: D96V + D111A + T231R + N233R, LVAR0074: N33Q + E87K +T231R + N233R, LVAR0076: N33Q + N94K + T231R + N233R, LVAR0077: N33Q +D96Y + T231R + N233R, LVAR0078: N33T + E43V + E56K + D96G + T231R +N233R, LVAR0079: N33Q + K98I + T231R + N233R, LVAR0080: A30V + N33Q +K98I + T231R + N233R, LVAR0086: N33Q + E87K + D96E + T231R + N233R,LVAR0088: N26I + N33Q + T231R + N233R, LVAR0091: A30T + N33Q + T231R +N233R, LVAR0094: N33Q + G91V + T231R + N233R, LVAR0095: N33Q + G91A +T231R + N233R, LVAR0096: N33Q + G91V + L97M + T231R + N233R, LVAR0099:N33Q + K98I + T231R + N233R, LVAR0101: N33Q + L69I + G91E + T231R +N233R, LVAR0102: P29T + N33Q + T231R + N233R, LVAR0103: N33Q + G91V +T231R + N233R, LVAR0104: N33Q + K98I + T231R + N233R, LVAR0106: N33Q +G91E + T231R + N233R, and LVAR0108: N33Q + N94K + T231R + N233R,wherein each position corresponds to a position of amino acids 1 to 269of SEQ ID NO: 2.

-   6. A lipase being a variant of a parent lipase, which variant (a)    has at least 50% identity to amino acids 1 to 269 of SEQ ID NO: 2;    and (b) has lipase activity; and (c) comprises at least one    substitution in at least one of positions 30, 42, 114, and/or 163,    wherein each position corresponds to a position of amino acids 1 to    269 of SEQ ID NO: 2.-   7. A lipase being a variant of a parent lipase, which variant (a)    has at least 50% identity to amino acids 1 to 269 of SEQ ID NO: 2;    and (b) has lipase activity; and (c) comprises at least one of the    following substitutions: D27Y, P29T, A30T, A30V, T32I, N33T, N33Y,    P42L, D57A, D57N, G91V, T114I, G163K, N251S, wherein each position    corresponds to a position of amino acids 1 to 269 of SEQ ID NO: 2.-   8. A lipase being a variant of a parent lipase, which variant (a)    has at least 50% identity to amino acids 1 to 269 of SEQ ID NO: 2;    and (b) has lipase activity; and (c) is selected from the following    variants:

LVA012: D27R + N33Q + G91A + D96E + L97Q + D111A + T231R + N233R +P256T, LVA023: N33Q + E210D + T231R + N233R, LVA041: N33Q + D111A +T231R + N233R, LVA043: N33Q + G91T + T231R + N233R, LVA049: N33Q +G163K + T231R + N233R, LVA061: D27Q + N33Q + T231R + N233R, LVA099:D27R + N33Q + G91T + N94S + D111A + S216P + L227G + T231R + N233R +P256T, LVA349: K98I + T231R + N233R + N251S, LV1232: G91A + D96W +E99K + G263Q + L264A + I265T + G266D + T267A + L269N, LV1330: N33Q +D96S + T231R + N233R + Q249R, LV1855: D27R + G91A + D111A + S216P +L227G + P256T, LV1857: D27R + G91N + N94R + D111A + S216P + L227G +P256T, LV1865: D27R + G91T + N94S + D111A + S216P + L227G + P256T,LV1874: D27R + G91S + D111A + S216P + L227G + P256T, LV1889: D27R +G91T + D96N + D111A + S216P + L227G + P256T, LVAR0002b T32I + G91V +T231R + N233R, LVAR0003: K98I + T231R + N233R, LVAR0011a G91A + T231R +N233R, LVAR0013: G91V + T231R + N233R, LVAR0014 N33Y + G91W + N94K +T231R + N233R, LVAR0015 P42L + D57N + G91E + T231R + N233R, LVAR0016K98I + T231R + N233R, LVAR0017 V60L + G91V + T231R + N233R, LVAR0032:D57G + L93F + T231R + N233R, LVAR0045: A49T + E56R + E87K + E99S +T231R + N233R, LVAR0046: E99T + T114I + D254N + T231R + N233R, LVAR0047:D27Y + E87K + D96L + E99P + T231R + N233R, LVAR0048 E43K + E56S + E87K +T231R + N233R, LVAR0050: E56S ++ E87K + D96L + E99D + T231R + N233R,LVAR0051: E56A + D57A + T114I + T231R + N233R, LVAR0052: G91E + T231R +N233R, LVAR0053: E56K + D96G + D111A + T231R + N233R, LVAR0054: E87K +D111S + T231R + N233R, LVAR0055 E43V + G91R + T231R + N233R, LVAR0056:E56S + E87K + T231R + N233R, LVAR0057: E87K + G91E + T231R + N233R,LVAR0058: D27Y + E87K + T231R + N233R, LVAR0059 E43M + E87K + D96L +E99P + T231R + N233R, LVAR0061: E56K + E87K + D111A + T231R + N233R,LVAR0062: E87K + E99P + T231R + N233R, LVAR0063: E87K + D96L + E99P +T231R + N233R, LVAR0064: E56C + E87K + T231R + N233R, LVAR0065: E56R +E87K + D96L + T231R + N233R, LVAR0066 E43D + E56A + D57A + E87K +D111A + T231R + N233R, LVAR0067: E56K + E87K + D96L + E99P + T231R +N233R, LVAR0068 E87K + L147S + T231R + N233R, LVAR0069: D27Y + E87K +D96L + E99P + T231R + N233R, LVAR0070 E43D + E87K + D96L + E99P +E239V + T231R + N233R, LVAR0071 E43K + E56A + E87K + D234K + T231R +N233R, LVAR0072: D96V + D111A + T231R + N233R, LVAR0074: N33Q + E87K +T231R + N233R, LVAR0076: N33Q + N94K + T231R + N233R, LVAR0077: N33Q +D96Y + T231R + N233R, LVAR0078: N33T + E43V + E56K + D96G + T231R +N233R, LVAR0079: N33Q + K98I + T231R + N233R, LVAR0080: A30V + N33Q +K98I + T231R + N233R, LVAR0086: N33Q + E87K + D96E + T231R + N233R,LVAR0088: N26I + N33Q + T231R + N233R, LVAR0091: A30T + N33Q + T231R +N233R, LVAR0094: N33Q + G91V + T231R + N233R, LVAR0095: N33Q + G91A +T231R + N233R, LVAR0096: N33Q + G91V + L97M + T231R + N233R, LVAR0099:N33Q + K98I + T231R + N233R, LVAR0101: N33Q + L69I + G91E + T231R +N233R, LVAR0102: P29T + N33Q + T231R + N233R, LVAR0103: N33Q + G91V +T231R + N233R, LVAR0104: N33Q + K98I + T231R + N233R, LVAR0106: N33Q +G91E + T231R + N233R, and LVAR0108: N33Q + N94K + T231R + N233R,wherein each position corresponds to a position of amino acids 1 to 269of SEQ ID NO: 2.

-   9. The lipase of any one of embodiments 1-8, in combination with a    protease or an amylase, for use as a medicament.-   10. The lipase of any one of embodiments 1-8, in combination with a    protease and an amylase, for use as a medicament.-   11. The lipase in combination with a protease and/or an amylase    according to embodiment 9 or 10, wherein (i) the protease has at    least 70% identity to a protease selected from the group consisting    of a) a protease having amino acids 1-274 of SEQ ID NO: 3, b) a    protease having amino acids 1-188 of SEQ ID NO: 4, and c) a protease    having amino acids 1-188 of SEQ ID NO: 5; (ii) the amylase has at    least 70% identity to an amylase selected from the group consisting    of a) an amylase having amino acids 1-481 of SEQ ID NO: 6, b) an    amylase having amino acids 1-481 of SEQ ID NO: 7, and c) an amylase    having amino acids 1-483 of SEQ ID NO: 8.-   12. Use of a lipase or a mixture of lipases as defined in any one of    embodiments 1-8 for the manufacture of a medicament for the    treatment of digestive disorders, pancreatic exocrine insufficiency,    pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type    II.-   13. The use of embodiment 12, further comprising the use of a    protease or an amylase.-   14. The use of embodiment 12, further comprising the use of a    protease and an amylase.-   15. The use of embodiment 13 or 14, wherein the protease and/or    amylase are as defined in embodiment 11.-   16. A lipase as defined in any one of embodiments 1-8 for use in the    treatment of digestive disorders, pancreatic exocrine insufficiency,    pancreatitis, cystic fibrosis, diabetes type I, and/or diabetes type    II.-   17. The lipase of embodiment 16, in combination with a protease or    an amylase.-   18. The lipase of embodiment 16, in combination with a protease and    an amylase.-   19. The lipase of embodiment 16 or 17, wherein the protease and/or    amylase are as defined in embodiment 11.-   20. A pharmaceutical composition comprising a lipase or a mixture of    lipases as defined in any one of embodiments 1-8, together with at    least one pharmaceutically acceptable auxiliary material.-   21. The composition of embodiment 20, further comprising a protease    or an amylase.-   22. The composition of embodiment 20, further comprising a protease    and an amylase.-   23. The composition of embodiment 21 or 22, wherein the protease    and/or amylase are as defined in embodiment 11.-   24. A method for the treatment of digestive disorders, pancreatic    exocrine insufficiency, pancreatitis, cystic fibrosis, diabetes type    I, and/or diabetes type II, by administering a therapeutically    effective amount of a lipase or a mixture of lipases as defined in    any one of embodiments 1-8.-   25. The method of embodiment 24, further comprising administering a    therapeutically effective amount of a protease or an amylase.-   26. The method of embodiment 24, further comprising administering a    therapeutically effective amount of a protease and an amylase.-   27. The method of embodiment 25 or 26, wherein the protease and/or    amylase are as defined in embodiment 11.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES

Chemicals used were commercial products of at least reagent grade.De-ionized water is from the Milli-Q system (QPAK1, Millipore, catalogueno. CPMQ004R1).

Example 1 Enzyme Assays

Assays for lipase, protease and amylase activity of porcine pancreatinhave been published by the FIP (Federation InternationalePharmaceutique) as well as the European Pharmacopoeia and the UnitedStates Pharmacopeia. 1 FIP-unit=1 Ph.Eur.-unit (European Pharmacopoeia).The assays are described in, e.g.: Fédération InternationalePharmaceutique, Scientific Section: International Commission for thestandardisation of pharmaceutical enzymes. a) “Pharmaceutical Enzymes,”Editors: R. Ruyssen and A. Lauwers, E. Story Scientia, Ghent, Belgium(1978), b) European Pharmacopoeia. See also Deemester et al in LauwersA, Scharpé S (eds): Pharmaceutical Enzymes, New York, Marcel Dekker,1997, p. 343-385. Appropriate enzyme standards can be procured from:International Commission on Pharmaceutical Enzymes, Centre forStandards, Harelbekestraat 72, B-9000 Ghent.

The lipase FIP assay as well as other suitable assays for lipase,protease and amylase is described below.

Ligase FIP Assay

For measuring lipolytic activity of pancreatin the method published inthe European Pharmacopoeia 5.1 was used. Unless otherwise stated, fordetermination of the lipolytic activity of microbial lipases the assayfor Rhizopus oryzae lipase published by the FIP was used.

Ligase pNP Assay

-   Substrate: para-Nitro-Phenyl (pNP) Valerate-   Assay pH: 7.7-   Assay temperature: 40° C.-   Reaction time: 25 min

The digested product with yellow colour has a characteristic absorbanceat 405 nm. Its quantity is determined by spectrophotometry. The lipaseactivity may be determined relative to an enzyme standard of knownactivity. The activity may be expressed in Lipolase Units (LU). One LU(Lipolase Unit) is the amount of enzyme which releases 1 mmol titratablebutyric acid per minute under the above standard conditions. 1 KLU=1000LU. A more detailed assay description, AF95/6-GB(Lipase/Esterase—pH-STAT Method on a Tributyrin Substrate (LU)), as wellas a LU standard, is available on request from Novozymes A/S,Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.

Lipase LU Assay

In this assay, the lipase-catalysed degradation of 0.16 M tributyrin(glycerol tributyrate, Merck 1.01958.000) at pH 7.00 and 30° C. (±1° C.)is followed by pH-stat titration of released butyric acid with 0.025 Mdegassed, CO₂-free sodium hydroxide (Sodium hydroxide titrisol, Merck9956). The consumption of the titrant is recorded as a function of time.

The substrate is emulsified with a 0.6% w/v Gum arabic emulsifier (20.0g Gum Arabic, 89.5 g NaCl, 2.05 g KH₂PO₄, add water to 1.5 l, leaveuntil completely dissolved, add 2700 ml glycerol, adjust pH to 4.5. 90ml of tributyrin is mixed with 300 ml gum arabic emulsifier and 1410 mldemineralised water and homogenised for 3 minutes using e.g. a Silversonemulsifier L4RT at 7000 rpm and then adjusted to pH 4.75).Lipase-samples are diluted first in 0.1 M glycin buffer pH 10.8, next indemineralized water, aiming at an activity level of 1.5-4.0 LU/ml. 15 mlof the emulsified substrate solution is poured into the titrationvessel. 1.0 ml sample solution is added, and pH is maintained at 7.0during the titration. The amount of titrant added per minute to maintaina constant pH is measured. The activity calculation is based on the meanslope of the linear range of the titration curve. A standard of knownactivity may be used as a level check.

1 LU (lipase unit) is the amount of enzyme which releases 1 micro moletitratable butyric acid per minute under the assay conditions givenabove. 1 kLU (kilo Lipase Unit)=1000 LU.

A more detailed assay description, EB-SM-0095.02, is available onrequest from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.

Lipase pH Stat Assay

This assay is based on the lipase-catalysed release of fatty acids froman olive oil emulsion in the presence of 0.65 mM bile salts. Thesubstrate is emulsified with gum arabic as emulsifier (175 g olive oilemulsified with 630 ml gum arabic solution (474.6 g gum arabic, 64 gcalcium chloride in 4000 ml water) for 15 min in a blender; aftercooling to room temperature, pH is adjusted to pH 6.8-7.0 using 4 MNaOH).

For the determination, 19 ml of the emulsion and 10 ml bile saltssolution (492 mg bile salts are dissolved in water and filled up to 500ml) are mixed in the reaction vessel and heated to 36.9° C. to 37.5° C.Reaction is started by addition of 1.0 ml of enzyme solution. Thereleased acid is titrated automatically at pH 7.0 by addition of 0.1 Msodium hydroxide for a total of 5 min. The activity is calculated fromthe slope of the titration curve between the 1st and the 5th minute. Forcalibration, a standard is measured at three different levels ofactivity.

Protease Suc-AAPF-pNA Assay

-   Substrate: Suc-AAPF-pNA (Sigma S-7388).-   Assay buffer: 100 mM succinic acid, 100 mM HEPES (Sigma H-3375), 100    mM CHES (Sigma C-2885), 100 mM CABS (Sigma C-5580), 1 mM CaCl₂, 150    mM KCl, 0.01% Triton-X100 (a nonionic surfactant having the    molecular formula: C₁₄H₂₂O(C₂H₄O)_(n) where the average number of    ethylene oxide units per molecule is around 9 or 10, CAS #:    9002-93-1) adjusted to pH 9.0 with HCl or NaOH.-   Assay temperature: 25° C.

300 microliters diluted protease sample was mixed with 1.5 ml of theassay buffer and the activity reaction was started by adding 1.5 ml pNAsubstrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with0.01% Triton-X100) and, after mixing, the increase in A₄₀₅ was monitoredby a spectrophotometer as a measurement of the protease activity. Theprotease samples were diluted prior to the activity measurement in orderto ensure that all activity measurements fell within the linear part ofthe dose-response curve for the assay.

Protease AU Assay

Denatured haemoglobin (0.65% (w/w) in urea-containing 6.7 mM KH₂PO₄/NaOHbuffer, pH 7.50) is degraded at 25° C. for 10 minutes by the proteaseand un-degraded haemoglobin is precipitated with trichloroacetic acid(TCA) and removed by filtration. The TCA-soluble haemoglobin degradationproducts in the filtrate are determined with Folin & Ciocalteu's phenolreagent (1 volume of Folin-Ciocalteu Phenol Reagent Merck 9001.0500 to 2volumes of demineralised water), which gives a blue colour with severalamino acids (being measured at 750 nm). The activity unit (AU) ismeasured and defined by reference to a standard. The denaturedhaemoglobin substrate may be prepared as follows: 1154 g urea(Harnstoff, Merck 8487) is dissolved in 1000 ml demineralised water,240.3 g NaOH is added and then, slowly, 63.45 g haemoglobin (Merck 4300)is added, followed by 315.6 g KH₂PO₄, and demineralised water ad 3260 g.pH is adjusted to 7.63. More details and a suitable Alcalase standardare available on request from Novozymes A/S, Krogshoejvej 36, DK-2880Bagsvaerd, Denmark (assay no. EB-SM-0349.01).

Amylase

-   Substrate: Phadebas tablets (Pharmacia Diagnostics; cross-linked,    insoluble, blue-coloured starch polymer, which is mixed with bovine    serum albumin and a buffer substance, and manufactured into tablets)-   Assay Temperature: 37° C.-   Assay pH: 4.3 (or 7.0, if desired)-   Reaction time: 20 min

After suspension in water the starch is hydrolyzed by the alpha-amylase,giving soluble blue fragments. The absorbance of the resulting bluesolution, measured at 620 nm, is a function of the alpha-amylaseactivity. The alpha-amylase activity may be determined relative to astandard of known activity, e.g. expressed in Fungal alpha-Amylase Units(FAU). One FAU is the amount of enzyme which breaks down 5.26 g starch(Merck, Amylum solubile Erg. B. 6, Batch 9947275) per hour at thestandard assay conditions. A more detailed assay description,APTSMYQI-3207, and a FAU standard, is available on request fromNovozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.

Example 2 Lipase Variants with Improved Phospholipase Activity

DNA encoding the lipase variants shown in Table 1 below was transformedinto Aspergillus oryzae strain ToC1512 (described in WO 2005/070962),using the method described in Example 22 of U.S. Pat. No. 5,869,438,except that PyrG selection was used (described in WO 2004/069872)instead of AMDS selection. Spores of the Aspergillus oryzae host weretaken from an agar slant and used for inoculation of 10 ml YPM (10 gyeast extract, Difco+20 g Peptone, Difco, water to 1 L, is autoclaved;add sterile filtered maltose to 2% (w/w)). Inoculated tubes wereincubated at 30° C. for three days in a New Brunswick Scientific Innova2300 shaker at 180 rpm. Supernatants were harvested by filteringcultures with Mira-Cloth (Calbiochem) followed by sterile filtrationwith 0.45 um (micro meter) filters. The lipase variants were purified asgenerally described in Example 23 of U.S. Pat. No. 5,869,438.

TABLE 1 Lipase variants I Variant designation Substitutions as comparedto SEQ ID NO: 2 LVA012 D27R + N33Q + G91A + D96E + L97Q + D111A +T231R + N233R + P256T LVA023 N33Q + E210D + T231R + N233R LVA041 N33Q +D111A + T231R + N233R LVA043 N33Q + G91T + T231R + N233R LVA049 N33Q +G163K + T231R + N233R LVA061 D27Q + N33Q + T231R + N233R LVA099 D27R +N33Q + G91T + N94S + D111A + S216P + L227G + T231R + N233R + P256TLVA103 Q4R + N33Q + T231R + N233R LVA120 N33Q + D96W + T231R + N233RLVA349 K98I + T231R + N233R + N251S LV1330 N33Q + D96S + T231R + N233R +Q249R LV1855 D27R + G91A + D111A + S216P + L227G + P256T LV1857 D27R +G91N + N94R + D111A + S216P + L227G + P256T LV1865 D27R + G91T + N94S +D111A + S216P + L227G + P256T LV1874 D27R + G91S + D111A + S216P +L227G + P256T LV1889 D27R + G91T + D96N + D111A + S216P + L227G + P256TLV2934 N33Q + T231R + N233R

The following lipases were used for comparison and were also prepared asdescribed above:

The wild type lipase from Humicola lanuginosa DSM 1800 having thesequence of amino acids 1-269 of SEQ ID NO: 2 and described forpharmaceutical use in, e.g., U.S. Pat. No. 5,614,189), and the(T231R+N233R)-variant thereof having amino acids 1-269 of SEQ ID NO: 1,described for pharmaceutical use in WO 2006/136159.

The following lipase serves as a positive control (positive forphospholipase activity):

Variant LV1232 with the following substitutions as compared to SEQ IDNO: 2: G91A+D96W+E99K+G263Q+L264A+1265T+G266D+T267A+L269N.

These lipases were tested for phospholipase activity as described in thefollowing. Enzymes:

The enzyme samples were diluted in enzyme dilution buffer (20 mMNa-Acetate, 0.01% w/w Triton-X100, pH 5.0) to 5 mg/mL (mg of enzymeprotein (EP) per ml). The enzyme concentrations were determined on thebasis of A₂₈₀ and the calculated molar absorption coefficient (programGPMAW (Lighthouse Data, Odense, Denmark; available on the Internet atwelcome.to/gpmaw; see also Gill and von Hippel, 1989, Calculation ofprotein extinction coefficients from amino acid sequence data, Anal.Biochem. 182: 319-326).

Substrate:

A solution of the substrate1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (in what follows“phosphatidylcholine”), which is commercially available from AvantiPolar Lipids Inc., 700 Industrial Park Drive, Alabaster, Ala. 35007, US,catalogue no. 850445) was prepared as follows:

-   1 137.5 mg phosphatidylcholine is dissolved in 750 uL (microliter)    de-ionized water-   2 Stir 1 h at room temperature-   3 Add 37.5 uL 0.32 M CaCl₂-   4 Stir for 1-2 minutes-   5 Add 375 uL 16 mM sodium deoxycholate-   6 Add 750 uL de-ionized water-   7 Stir for 30 minutes at room temperature    Enzyme Reaction:-   1 Transfer 100 uL substrate to 2 mL-eppendorf tubes-   2 Add 5 uL enzyme, diluted to 5 mg/mL as described above-   3 Incubate for 20 minutes at 40° C., 1000 rpm in an eppendorf    thermomixer-   4 Transfer 10 uL reaction mixture to a new eppendorf tube and add    990 uL 50% methanol (MeOH), 0.1% (w/w) trifluoroacetic acid (TFA)-   5 This is analysed by MALDI-TOF MS (Matrix-Assisted Laser    Desorption/Ionization Time-Of-Flight mass spectrometry), after    mixing with this matrix: 20 mg/mL 2,5-dihydrobenzoic acid in 50%    MeOH, 0.1% TFA

A substrate control was included, in which 5 uL enzyme dilution bufferwas added instead of enzyme in step 2. Four independent determinationswere made for each sample in step 5. The MALDI-TOF MS apparatus used wasa Voyager DE PRO instrument with positive ionisation in reflector modewith external calibration (Calmix 2, Applied Biosystems).

By choosing a glycerol-based substrate with ester-bonded acids ofdifferent length it is possible to distinguish between enzymaticspecificities (attack at the 1-position or 2-position) by measuring themass of the digested glycerol backbone. The masses of the variouspossible digested glycerol backbones are:

706 Da Phosphatidylcholine 496 Da A1 hydrolysis (phosphatidylcholineminus myristoyl (C14) in position 1) 468 Da A2 hydrolysis(phosphatidylcholine minus palmitoyl (C16) in position 2) 258 Da A1 andA2 hydrolysis (undegraded phosphatidylcholine minus C14 and C16)Results:

The relative signal intensities (area under each peak) of the MS peaksrepresenting Mw's of 706, 496, 468, and 258Da are used as basis for thecalculation of the distribution between Phospholipase A1 and A2 (PLA1-and PLA2-) activity.

The results, from two different experiments (I and II), are shown inTables 2 and 3 below.

Generally, a signal intensity of above 10-15% of A1/A2 relative to A2/A1may indicate either true dual activity or an impure sample.

TABLE 2 Experiment I: Phospholipase activity of lipase variants %undigested Enzyme Distribution of activity phospholipid tested PLA-1 %PLA-2 % left after hydrolysis SEQ ID NO: 1 72 28 44 SEQ ID NO: 2 78 2242 LVA012 66 34 65 LVA023 78 22 40 LVA041 79 21 55 LVA061 73 27 46LVA099 71 29 45 LVA103 69 31 68 LVA349 80 20 46 LVA120 75 25 47 LV188963 37 26 LV2934 60 40 62 LV1232 68 32 1.4 Substrate 82 control

TABLE 3 Experiment II: Phospholipase activity of lipase variants %undigested Enzyme Distribution of activity phospholipid tested PLA-1 %PLA-2 % left after hydrolysis SEQ ID NO: 1 73 27 82 LV1330 86 15 54LV1855 84 16 58 LV1865 78 22 65 LV1874 84 17 56 LV1889 89 11 4.6 LVA04382 18 51 LVA049 82 18 50 LV1857 86 14 42 LV1232 91 9.3 1.3 Substrate 100controlConclusion:

All lipases tested have phospholipase activity to some extent, primarilyas PLA1-activity. The positive control, LV1232, showed a highphospholipase activity in both experiments.

The prior art lipases of SEQ ID NOS: 1 and 2 showed almost the sameperformance as regards phospholipase activity when tested in the sameexperiment (Experiment I), leaving approximately 44% and 42%,respectively, undigested phospholipid after hydrolysis.

It is contemplated that the following lipase variants have an improvedphospholipase activity as compared to SEQ ID NO: 2: LV1232, LV1889 andLVA023 (Experiment I), and LV1232, LV1330, LV1855, LV1865, LV1874,LV1889, LVA043, LVA049, and LV1857 (Experiment II). Variant LV1232 andLV1889 in particular show a very much improved phospholipase activity inboth experiments.

Example 3 Lipase Variants with Improved Activity at pH 6

A number of the purified lipase variants shown in Table 1 above weretested for activity at pH 6 in the presence of 10 mM bile salt. Like inExample 1, the lipases of SEQ ID NO: 2 and 1 were included forcomparison.

Chemicals and Reagents:

-   Assay buffer pH 6: 100 mM imidazole, 100 mM acetate, 100 mM malonic    acid, pH 6.0-   Enzyme dilution buffer: 5 mM NaH₂PO₄ pH 7.0-   7 mM CaCl₂ (Merck, 1.02382.0500)-   Bile salts (80 mM): Lipase activating bile salt mixture from Solvay    Pharmaceuticals, batch 176.01-PA-7374-   Trilinolein (glyceryl trilinoleate, Sigma T9517)-   Pepsin: (Merck VL 317492437, catalogue no. 1.0792.0001)-   Stop solution: 10% Triton-X100, 1 M Phosphoric acid.    Substrate:-   Substrate working emulsion was prepared as followed:-   1. Mix 2.188 mL bile salts (80 mM) with 6.68 mL de-ionized water.-   2. Add 0.133 mL Trilinolein (glyceryl trilinoleate, Sigma T9517).-   3. Mix 1 minute with ultraturex mixer (yellow line DI 25 basic) at    room temperature.-   This gives a working emulsion of substrate with 19.44 mM bile salts    and 15.56 mM Trilinolein.    Enzymes:

The enzyme samples were diluted in 5 mM NaH₂PO₄ pH 7.0 to 0.07 mg/mL (mgof enzyme protein per ml). The enzyme concentrations were based on A₂₈₀.Enzymes were two-fold diluted in 5 mM NaH₂PO₄ (six dilutions made intotal and no enzyme/buffer control). These dilutions give the followingfinal concentrations of enzyme in the wells: 0.01 mg/ml, 0.05, 0.025,0.0125, 0.0625, 0.03125, and 0.015625 mg/ml.

Assay Procedure:

-   1. Mix in Micro Titer Plates (MTP) 35 ul (micro liter) assay buffer    with 25 ul 7 mM CaCl₂. Add 90 ul substrate working emulsion.    Pre-incubate for 20 min at 37° C., 700 rpm.-   2. Add 25 ul of the respective enzyme dilutions, and incubate 30 min    at 37° C., 700 rpm (final volume 175 ul). Final concentration of    bile salts and trilinolein is 10 mM and 8 mM, respectively, in MTP    well.-   3. Add 50 ul stop solution (10% Triton-X100, 1 M Phosphoric acid)    and 25 ul pepsin (700 mg/l). Incubate 10 min at room temperature.    The pepsin, which is a protease, is added in order to avoid    re-activation of the lipase protein when the pH is increased in the    subsequent procedure (the determination of free fatty acids (FFA)).-   4. Dilute samples immediately 10 times in 1% Triton-X100 for    detection of FFA by NefaC (Wako, Nefa C ACS-ACOD Method Enzymatic    color test Code No: 999-75406).    Detection of free fatty acids by NefaC kit (Nefa C ACS-ACOD Method    Enzymatic color test Code No: 999-75406):-   1. Make solution A: Bottle R1a is dissolved with 10 ml of bottle R1    (from NefaC kit)

Make solution B: Bottle R2a is dissolved with 20 ml of bottle R2 (fromNefaC kit)

-   2. Cal1 Nefa C standard Oleic acid (28.2 mg/dl-1 mmol/L) is diluted    in 1% Triton-X100 to obtain the following concentrations for NefaC    standard: 1 mM, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625 mM.-   3. 25 ul standard/lipase sample is mixed with 50 ul solution A.    Incubate 15 min at room temperature, 700 rpm.-   4. Add 100 ul solution B, incubation 15 min room temperature, 700    rpm.    Results:

The concentration of FFA in mM is determined from the Nefa C standardcurve. The lipase activity results are fitted to Michealis-Menten-likefit:A=A0+Amax*[E]/([E]+K)

V0 is determined (mmol FFA/g enzyme/min), and the ratio to the V0 forthe lipase of SEQ ID NO: 1 is determined. The lipase of SEQ ID NO: 1 andvariant LV2934 (a non-glycosylated variant of SEQ ID NO: 1) wereincluded on each MTP as controls.

The results, normalized to SEQ ID NO: 1, are shown in Table 4 below.

TABLE 4 Lipase activity at pH 6 on trilinoleate Enzyme tested RelativeV0 (mmol FFA/g enzyme/min) SEQ ID NO: 2 0.35 LVA049 1.45 LVA349 1.10LVA023 1.06 LVA099 1.02 SEQ ID NO: 1 (I) 1.00 SEQ ID NO: 1 (II) 1.00 SEQID NO: 1 (III) 1.00 LVA061 0.90 LV2934-I 0.80 LV2934-II 0.77 LV2934-III0.73 LV1330 0.79 LVA043 0.75 LVA041 0.72 LVA012 0.64 LV1857 0.47 LV18550.36 LV1889 0.34 LV1874 0.33 LV1865 0.23Conclusion:

On the basis of the above results it is contemplated that, except forLV1889, LV1874 and LV1865, all variants tested have a higher activity atpH 6 using trilinoleate substrate with 10 mM bile salts, as compared tothe lipase of SEQ ID NO: 2.

The LVA049, LVA349, LVA023, and LVA099 lipase variants seem much betterthan the comparative lipase of SEQ ID NO: 2 in this respect, in factbetter than the SEQ ID NO: 1 lipase. This is so in particular for theLVA049 and LVA349 lipase variants, more in particular for the LVA049lipase variant.

Example 4 Lipase Variants with Improved Stability at pH 3

A number of the lipase variants listed in Table 1 above were tested forpH stability in the pH range of 2-8. Like in the previous Examples, thelipases of SEQ ID NO: 2 and 1 were included for comparison. Thefollowing variants of the lipase of SEQ ID NO: 2 were also tested:

LVA147: D27R + N33Q + G91N + N94R + D111A + S216P + L227G + T231R +N233R + P256T LVA315: N33Q + G91T + G163K + T231R + N233R + D254GLVA317: N33Q + G91T + G163K + T231R + N233R + D254S LVA319: N11R +N33Q + G91T + G163K + T231R + N233R + D254S LVA714: D27V + N33Q + G91A +N94R + D111A + G163K + L227F + T231R + N233R + Q249R + D254S

Each enzyme was tested in duplicate, in two concentrations (0.05 and 1.0mg enzyme protein/ml). In addition, the enzymes were tested with andwithout bile salts 10 mM, and with and without pepsin (70 mg/l).

In brief, the enzymes were incubated at 37° C. at the desired pH for 1,15, 45, and 120 minutes (or for 1, 60, and 120 minutes), following whichresidual lipase activity was measured on p-Nitrophenyl caprylate at pH 8and room temperature (RT).

Chemicals/Reagents:

-   Enzyme dilution buffer: 20 mM acetate pH 6, 0.01% Triton-X100.-   Stability buffer: 200 mM imidazole, 200 mM acetate, 200 mM malonic    acid, adjusted to pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0.-   Residual activity buffer (RA buffer): 200 mM Tris    (tris-hydroxymethyl aminomethan,    2-amino-2-hydroxymethyl-1,3-propandiol, CAS-number: 77-86-1) pH 8,    0.4% Triton-X100, 1 mM CaCl₂-   pNP-caprylate (C8): Sigma N-0752-   Pepsin (700 mg/I): Merck, VL 317492437 (1.0792.0001)-   Bile salts (80 mM): Lipase activating bile salt mixture from Solvay    Pharmaceuticals (batch 176.01-PA-7374)    Enzymes:

Enzymes were diluted in 20 mM NaH₂PO₄ pH 7.0, 0.01% Triton-X100 toworking solutions of 0.4 or 0.8 mg enzyme protein per ml, based on A₂₈₀.

Stability Assay:

-   1) To each well of a microtiter plate (MTP) add 50 ul stability    buffer, 20 ul 0.1% Triton-X100, 25 ul bile salts (80 mM) or 20 ul    pepsin (700 mg/l) or de-ionized water. Add de-ionized water to a    final volume of 175 ul per well. All samples are made in duplicate.    Preheat at 37° C. for 20 min, 700 rpm.-   2) Add 25 ul enzyme (final concentration 0.05 or 0.1 mg enzyme    protein per ml). For each pH no enzyme controls with pepsin and bile    salts are included, ie. 25 ul 20 mM NaH₂PO₄pH 7.0, 0.01% Triton-X100    is added to these wells. Incubate at 37° C. 700 rpm for 1, 15, 45    and 120 min.-   3) Withdraw aliquots of 20 ul and dilute sample with 180 ul residual    activity buffer pH 8.0, keep samples on ice.-   4) Dilute sample (minimum dilution 20× to increase pH to 8). Make    initial residual activity (RA) on 1 min samples pH 8, diluted 20×,    40×, 80×, 160×, and 320× to determine dilution required to obtain    linearity in assay.-   5) Measure RA with pNP-caprylate substrate at pH 8.0 as described    below.    Substrate:

Substrate stock solution is prepared by mixing 14.2 ul p-Nitrophenylcaprylate (Sigma N-0752) with 1 ml 2-propanol. This stock solution isdiluted 50× in residual activity buffer pH8.0 giving a working solutionof which 150 ul is added to each well.

Procedure for Determining Residual Activity (RA):

-   1) Mix in new MTP 20 ul diluted sample and 150 ul substrate working    solution.-   2) Measure kinetics at 405 nm for 5 min (mix first time, read every    12 sec, room temperature).    Results:

The % residual activity is calculated as follows: The rate within eachpH for each withdraw (1, 15, 45, 120 minutes) is subtracted the rate forno enzyme control with bile salts or pepsin. This corrected rate is thendivided by the highest value within each pH and multiplied by 100.

Table 5 below shows the stability at pH 3 for those variants that areimproved as compared to SEQ ID NO: 2 (in buffer, in the presence ofpepsin, or in the presence of bile salts, respectively). Only thestability results at pH 3 are shown, as the most pronounced differenceswere observed at this pH.

TABLE 5 Lipase variant residual activity after pre-incubation at pH 3Time Time Lipase Std. dev. Buffer Pepsin Bile salts Lipase Std. dev.Buffer Pepsin Bile salts SEQ ID NO: 2  1 min 100.0 100.0 100.0 LVA049  1min 100.0 std dev 2.6 9.6 1.1 std dev 4.9 15 min 82.6 2.1 5.2 15 min92.0 std dev 1.2 0.7 4.6 std dev 5.3 45 min 80.9 1.6 2.0 45 min 85.4 stddev 2.9 0.8 1.7 std dev 5.8 120 min  59.0 −1.2 −0.2 120 min  74.6 stddev 4.9 1.2 0.3 std dev 7.1 SEQ ID NO: 1  1 min 100.0 100.0 100.0 LV1855 1 min 100.0 100.0 std dev 3.1 2.2 6.5 std dev 0.7 2.4 15 min 72.2 1.139.2 15 min 92.1 90.4 std dev 0.5 0.1 0.2 std dev 1.3 0.6 45 min 62.20.1 20.5 45 min 90.6 67.5 std dev 0.3 0.0 3.1 std dev 0.0 3.2 120 min 59.2 0.0 10.5 120 min  88.4 31.5 std dev 1.3 0.0 1.4 std dev 3.1 2.2LV2934  1 min 100.0 LV1865  1 min 100.0 100.0 100.0 std dev 2.5 std dev3.8 0.4 0.7 15 min 84.9 15 min 92.5 85.0 50.2 std dev 0.5 std dev 0.40.9 1.3 45 min 73.9 45 min 92.8 66.5 36.4 std dev 0.4 std dev 0.1 1.10.4 120 min  62.6 120 min  92.7 30.3 14.6 std dev 1.1 std dev 0.7 0.51.3 LVA043  1 min 100.0 100.0 LV1874  1 min 100.0 100.0 std dev 3.4 1.5std dev 7.0 3.8 15 min 91.2 51.0 15 min 84.9 72.3 std dev 2.7 9.6 stddev 1.4 5.1 45 min 91.7 34.3 45 min 89.7 60.5 std dev 3.4 6.1 std dev2.6 1.9 120 min  83.4 25.1 120 min  87.9 23.6 std dev 3.1 5.1 std dev1.7 1.9 LV1889  1 min 100.0 100.0 LVA041  1 min 100.0 std dev 5.2 3.1std dev 3.0 15 min 97.4 68.2 15 min 85.3 std dev 6.0 6.2 std dev 2.9 45min 92.8 36.0 45 min 80.1 std dev 9.4 5.4 std dev 3.7 120 min  88.0 7.8120 min  69.6 std dev 5.7 2.8 std dev 5.9 LV1857  1 min 100.0 100.0LVA061  1 min 100.0 std dev 4.0 5.3 std dev 3.1 15 min 95.8 80.3 15 min87.8 std dev 0.7 2.2 std dev 2.9 45 min 94.5 54.2 45 min 83.0 std dev1.8 2.4 std dev 7.0 120 min  72.4 17.8 120 min  77.6 std dev 3.2 1.1 stddev 7.2 LVA012  1 min 100.0 100.0 LVA099  1 min 100.0 100.0 std dev 6.01.4 std dev 4.0 2.8 15 min 98.3 83.1 15 min 93.3 93.8 std dev 6.4 1.0std dev 3.7 1.4 45 min 88.2 54.8 45 min 89.9 95.2 std dev 4.6 1.3 stddev 3.4 1.2 120 min  78.4 20.1 120 min  74.7 94.0 std dev 5.5 1.7 stddev 9.0 2.4 LVA023  1 min 100.0 LVA349  1 min 100.0 std dev 3.1 std dev8.2 15 min 94.7 15 min 44.2 std dev 5.2 std dev 12.2 45 min 88.8 45 min24.2 std dev 5.5 std dev 13.3 120 min  83.7 120 min  31.2 std dev 7.2std dev 18.3 LVA147  1 min 100.0 LVA714  1 min 100.0 100.0 std dev 2.2std dev 2.9 0.9 60 min 20.1 60 min 89.1 70.9 std dev 1.4 std dev 0.5 3.3120 min  4.7 120 min  84.7 54.4 std dev 0.4 std dev 0.5 3.6 LVA319  1min 100.0 std dev 6.3 15 min 68.4 std dev 8.2 45 min 28.5 std dev 11.2120 min  6.3 std dev 4.8 LVA315  1 min 100.0 LVA317  1 min 100.0 std dev4.8 std dev 3.2 15 min 27.2 15 min 63.5 std dev 9.1 std dev 6.1 45 min5.3 45 min 27.5 std dev 5.0 std dev 9.5 120 min  0.9 120 min  3.6 stddev 1.0 std dev 2.5Conclusion:

The following lipase variants have an improved stability at pH 3,compared to the lipase of SEQ ID NO: 2: LV2934, LVA043, LVA049, LV1855,LV1865, LV1874, LV1889, LV1857, LVA012, LVA023, LVA041, LVA061, LVA099,LVA147, and LVA714.

The following lipase variants have an improved stability at pH 3 withpepsin, compared to the lipase of SEQ ID NO: 2: LVA043, LV1855, LV1865,LV1874, LV1889, LV1857, LVA012, LVA099, LVA315, LVA317, LVA319, andLVA714.

The following variants have an improved stability at pH 3 with bilesalts, compared to the lipase of SEQ ID NO: 2: LVA349.

Example 5 Micro-Purification of Lipase Variants

Additional lipase variants shown in Table 6 below were prepared asdescribed in Example 2, up to and including the sterile filtration ofthe fermentation supernatant.

TABLE 6 Lipase variants II Variant designation Substitutions as comparedto SEQ ID NO: 2 LVAR0003 K98I + T231R + N233R LVAR0013 G91V + T231R +N233R LVAR0032 D57G + L93F + T231R + N233R LVAR0045 A49T + E56R + E87K +E99S + T231R + N233R LVAR0046 E99T + T114I + D254N + T231R + N233RLVAR0047 D27Y + E87K + D96L + E99P + T231R + N233R LVAR0050 E56S +E87K + D96L + E99D + T231R + N233R LVAR0051 E56A + D57A + T114I +T231R + N233R LVAR0052 G91E + T231R + N233R LVAR0053 E56K + D96G +D111A + T231R + N233R LVAR0054 E87K + D111S + T231R + N233R LVAR0056E56S + E87K + T231R + N233R LVAR0057 E87K + G91E + T231R + N233RLVAR0058 D27Y + E87K + T231R + N233R LVAR0061 E56K + E87K + D111A +T231R + N233R LVAR0062 E87K + E99P + T231R + N233R LVAR0063 E87K +D96L + E99P + T231R + N233R LVAR0064 E56C + E87K + T231R + N233RLVAR0065 E56R + E87K + D96L + T231R + N233R LVAR0067 E56K + E87K +D96L + E99P + T231R + N233R LVAR0069 D27Y + E87K + D96L + E99P + T231R +N233R LVAR0072 D96V + D111A + T231R + N233R LVAR0074 N33Q + E87K +T231R + N233R LVAR0076 N33Q + N94K + T231R + N233R LVAR0077 N33Q +D96Y + T231R + N233R LVAR0078 N33T + E43V + E56K + D96G + T231R + N233RLVAR0079 N33Q + K98I + T231R + N233R LVAR0080 A30V + N33Q + K98I +T231R + N233R LVAR0086 N33Q + E87K + D96E + T231R + N233R LVAR0088N26I + N33Q + T231R + N233R LVAR0091 A30T + N33Q + T231R + N233RLVAR0094 N33Q + G91V + T231R + N233R LVAR0095 N33Q + G91A + T231R +N233R LVAR0096 N33Q + G91V + L97M + T231R + N233R LVAR0099 N33Q + K98I +T231R + N233R LVAR0101 N33Q + L69I + G91E + T231R + N233R LVAR0102P29T + N33Q + T231R + N233R LVAR0103 N33Q + G91V + T231R + N233RLVAR0104 N33Q + K98I + T231R + N233R LVAR0106 N33Q + G91E + T231R +N233R LVAR0108 N33Q + N94K + T231R + N233R

The Sterile filtered lipase-containing culture supernatants weremicro-purified using the following protocol:

To the wells of a filter plate (Unifilter 800, 25 um (micro meter) meltblown polypropylene filter, Whatman) approximately 50 ul (micro liter)XpressLine ProA chromatographic medium was added (commercially availablefrom Upfront Chromatography A/S, Lersoe Parkalle 42, DK-2100 Copenhagen,Denmark). The chromatographic medium was equilibrated by adding 200 ul 1M sodium acetate, pH 5.0. After 10 min agitation the equilibrationbuffer was removed by vacuum (Uni-Vac 3, Whatman). The equilibrationstep was repeated. Then 100 ul binding buffer (1 M sodium acetate, pH5.0) and 400 ul culture supernatant were added and mixed with thechromatographic medium for 30 min. Non-bound material was removed byvacuum. The binding step was repeated. The chromatographic medium withbound lipase was washed 3 times with 200 ul washing buffer withdecreasing buffer capacity (100/50/10 mM sodium acetate, pH 5.0). Ineach washing step the buffer was added, the plate was agitated 10 min,and the washing buffer was removed by vacuum. Finally, the bound lipasewas eluted by adding two times 100 ul 100 mM Tris, 0.02% Brij 35(Polyoxyethylen(23)laurylether), pH 9.0. For each elution step the platewas agitated 15 min before the eluted lipase was collected in amicrotiter plate by vacuum.

Example 6 Determination of Concentration of Lipase Variants

Active Site Titration

The concentration of lipase variants which had been purified(conventionally purified as generally described in Example 2, and/ormicro-purified as described in Example 6) were determined by burstactive site titration as described in the following.

The purified lipase was diluted in 0.01% Triton-X100, if necessary, toget concentration below 5 uM (corresponding to 150 ug enzymeprotein/ml). 100 ul purified lipase was mixed with approximately 100 ulof 40 uM of resorufin (ethyl resorufinyl heptylphosphonate; a lipaseinhibitor) dissolved in 1 M Tris, 4 mM SDS (Sodium Dodecyl Sulphate), pH9.0 in the well of a black microtiter plate. The precise concentrationof resorufin is not important, it only has to be added in excess ascompared to the 5 uM of lipase. Immediately after mixing, kinetics offluorescence from liberated resorufin was measured every minute for 1-3hours (until bursts were finalized) (excitation at 515 nm, emission at590 nm, measured on a POLARstar fluorescence intensity measuringinstrument from BMG LabTechnologies GmbH).

Measured fluorescence values are fitted to the equation:F=F0+Burst*(1−exp(−(t+dt)*ln(2)/T½)+Slope*(t+dt)where F is the measured fluorescence, F0 is the fluorescence backgroundfrom inhibitor and lipase, t is the time since first fluorescencemeasurement, dt is the time from mixing of lipase with inhibitor to thefirst fluorescence measurement, Burst is the fluorescence burst, T½ isthe half-time for the exponential burst, and Slope is the slope for thelinear change in fluorescence, e.g. due to hydrolysis of lipase-ethylheptylphosphonate complex and/or bleaching of resorufin.

The active lipase concentration was determined as the ratio between thecalculated burst and the slope of a resorufin standard curve (0-4 uM;included on the microtiter plate).

Concentration Determination from A₂₈₀

The concentration of the purified lipase variants was also estimatedfrom the absorbance at 280 nm using the extinction coefficient 1.24A₂₈₀/mg.

Example 7 In vitro Test of Lipase Variants

The purified lipase variants were tested in an in vitro digestion modelas described below.

Either of two diets (Diet I, and Diet II, respectively) was used in thein vitro model. The composition of Diet I is 34% (w/w) fat, 45% (w/w)carbohydrate, 2% (w/w) protein (the remainder water, salts, etc.). Thecomposition of Diet II is: Fat 313, protein 146, and starch 358(Nitrogen free Extract, NfE, may be calculated to 432), all in g/kg dryweight.

Diet I was prepared by mixing 247.2 g cow's milk (1.5% fat), 29.9 golive oil, 87 g Calshake (commercially available from Fresenius Kabi andhaving an energy content of 2077 kJ/g, a protein content of 4.3 g milkprotein/100 g, and a fat content of 24.4 g fat/100 g), and 9.9 gMethocel (Food Grade, E5 Premium LV FG (E464); Dow) using an UltraTurrex(YellowLine DI 25 basic) for 2 minutes. To reduce viscosity the diet wastreated with 0.5 ug/ml of the SAVINASE 16.0 LEX protease (commerciallyavailable from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd,Denmark) at pH 8.0 for 4 hours at 50° C. The protease was theninactivated by reducing pH to 3 and incubating at 70° C. for 30 min, or50° C. for 60 min.

Diet II consists of 73 g/kg (wet weight) poultry meal (Altromin), 73g/kg pea meal, 73 g/kg casein (precipitated under acidic conditions,from Altromin), 290 g/kg wheat flour, 290 g/kg potato starch, 125 g/kglard, 76 g/kg vitamins, minerals and trace elements, 375 g/kg cow'scream (33% fat).

100 ul of diet was mixed with 20 ul pepsin (Merck VL 317492437,catalogue no. 1.0792.0001, 700 mg/ml) and 30 ul lipase (duplicate of 4concentrations) in the well of a microtiter plate. If the gastric stepwas to be run at pH 5, 10 ul buffer (0.8 M MES(2-[N-morpholino]ethanesulfonic acid), 0.8 M sodium acetate, 0.8 Mimidazole, pH 7.0) was added, whereas no buffer was added if pH 3 wasused for gastric step. The microtiter plate was incubated 1 hour at 37°C. with shaking (Eppendorf Thermomixer, 750 rpm) before adding 15 ul (ifpH 5 used for gastric step), or 25 ul (if pH 3 used for gastric step)buffer (0.8 M MES (2-[N-morpholino]ethanesulfonic acid), 0.8 M sodiumacetate, 0.8 M imidazole, pH 7.0) and 20 ul bile salts (50 g/l,corresponding to 100 mM, using an average molecular weight of 500g/mol); Lipase activating bile salt mixture from Solvay Pharmaceuticals(batch 176.01-PA-7374)) resulting in a pH of 5.7 to 6.0. The plate wasthen incubated 2 hours at 37° C. with agitation before stopping thereaction by adding 50 ul 10% Triton-X100 in 1 M phosphoric acid. Afterdiluting 125-250 times in 1% Triton-X100 the amount of free fatty acidswas determined using a NEFA C kit from Wako Chemicals, as described inExample 3.

The dose response curves are fitted to the equation:FFA=FFAmax*[E]/([E]+K)where FFA is the amount of released free fatty acids, FFAmax is themaximal amount of free fatty acids that the lipases can liberate fromthe diet, [E] is the lipase concentration, and K is the lipaseconcentration that liberates half of FFAmax. Assuming that FFAmax isidentical for the lipases an improvement factor (IF) is defined as:IF=K(ref)/K(lipase)where K(ref) is the concentration of a reference lipase that liberateshalf of FFAmax and K(lipase) is the lipase variant concentration thatliberates half of FFAmax.

For the variants listed in Tables 7a and 8a below, as a reference lipasewe have used the average of the lipase of SEQ ID NO: 1 and itsdeglycosylated variant N33Q (LV2934 in Table 1), i.e., K(ref)=½×(K(SEQID NO: 1)+K(LV2934)).

The lipase variants listed in Tables 7a and 8a below all have animprovement factor above 1.0. This means that a lower amount of theselipases is required in order to obtain a similar effect as compared tothe reference lipase. The improvement factor of any lipase variantrelative to, e.g., SEQ ID NO: 2 can be calculated as the improvementfactor of the lipase variant in question relative to the referencelipase divided by the (constant) improvement factor of SEQ ID NO: 2relative to the reference lipase, which is indicated in Table 7a below.When for example Active Site Titration (AST, Example 6) is used todetermine the lipase concentration, and if the improvement factor forthe variant in question is to be calculated relative to the lipase ofSEQ ID NO: 2, one divides the average IF of the variant in question bythe average IF of SEQ ID NO: 2 which is 0.88, preferably 0.9.

For the additional lipase variants listed in Tables 7b, 8b, 8c, and 8dbelow, the deglycosylated variant N33Q of SEQ ID NO: 1 (LV2934 inTable 1) was used as reference lipase. Each of these lipases have animprovement factor above 1.00 (average improvement factor minus thestandard deviation). For the selection of the improved lipases, IFvalues and standard deviations were used with two decimals. Thesefigures were subsequently rounded to one decimal.

TABLE 7a Lipase variants with an improved performance in vitro(micro-purified) Improvement Factor (IF) Enzyme tested AST A₂₈₀ Diet I(pH 3 in gastric step) SEQ ID NO: 2 0.9 +/− 0.1 0.9 +/− 0.2 LVAR0003 1.9+/− 0.4 2.4, 2.1 +/− 0.4 LVAR0045 4.1 — LVAR0046 5.5 — LVAR0047 4.3 —LVAR0050 4.1 — LVAR0051 1.8 — LVAR0052 3.2 — LVAR0053 3.5 — LVAR0054 3.8— LVAR0056 2.2 +/− 0.6 — LVAR0057 3.0 — LVAR0061 2.3 — LVAR0062 2.4 —LVAR0063 6.6 — LVAR0064 2.0 +/− 0.0 — LVAR0065 4.6 — LVAR0067 8.0 —LVAR0069 7.3 — LVAR0072 2.5 — Diet II (pH 3 in gastric step) LVAR00743.3 2.2 LVAR0076 5.0 3.2 LVAR0077 4.5 2.5 LVAR0078 2.9 1.5 LVAR0079 6.77.7 LVAR0080 7.1 5.9 LVAR0086 4.3 2.7 LVAR0088 1.8 3.1 LVAR0091 5.2 4.3LVAR0094 11.0 10.5 LVAR0095 9.3 11.5 LVAR0096 8.3 6.6 LVAR0099 11.1 11.8LVAR0101 8.1 8.2 LVAR0102 6.0 4.1 LVAR0103 7.5 4.3 LVAR0104 7.9 5.9LVAR0106 10.0 9.7 LVAR0108 6.1 6.5 — means “not determined”

TABLE 7b Additional lipase variants with an improved performance invitro Lipase Substitutions as compared to SEQ ID NO: 2 IF (AST) Diet IpH 3 in gastric step LVA129 D27V + N33Q + V60S + D96W + T231R + 5.7 +/−2.0 N233R + Q249R LVA130 D27V + N33Q + V60S + T231R + N233R + 1.5 +/−0.4 Q249R LVA139 Q9H + N33Q + D102E + T231R + N233R 2.4 +/− 1.2 LVA140N33Q + D111E + T231R + N233R 1.8 +/− 0.3 LVA143 N33Q + D122E + T231R +N233R 1.8 +/− 0.5 LVA147 D27R + N33Q + G91N + N94R + D111A + 5.6 +/− 3.4S216P + L227G + T231R + N233R + P256T LVA180 N33Q + T231R + N233R +P256T 2.3 +/− 0.7 LVA182 D27R + N33Q + G91A + L93* + N94* + 1.2 +/− 0.0F95* + D96* + D111A + T231R + N233R + P256T LVA185 N11R + N33Q + T231R +N233R 1.9 +/− 0.7 LVA198 N33Q + N39H + T231R + N233R 1.4 +/− 0.3 LVA202N33Q + P229R + T231R + N233R 1.5 +/− 0.5 LVA206 D27R + N33Q + G91N +N94R + D111A + 2.9 +/− 0.8 G163K + S216P + L227G + T231R + N233R + P256TLVA208 N33Q + G91T + G163K + T231R + N233R 4.6 +/− 2   LVA210 D27R +N33Q + G91A + D96E + L97Q + 1.3 +/− 0.3 D111A + S216P + L227G + T231R +N233R + P256T LVA211 D27R + N33Q + G91A + D96E + L97Q + 4.3 +/− 1.2D111A + S216P + T231R + N233R + P256T LVA238 D27R + N33Q + G91A + D96E +D111A + 3.8 T231R + N233R + D254G + P256T LVA241 D27R + N33Q + G91A +N94S + D111A + 1.8 T231R + N233R + P256T LVA243 N33Q + N200S + T231R +N233R 1.9 +/− 0.3 LVA245 N33Q + N39S + T231R + N233R 3.6 +/− 0.0 LVA247N33Q + E210R + T231R + N233R 3.3 +/− 0.6 LVA248 N33Q + N39H + T231R +N233R + D254R 2.4 +/− 0.9 LVA249 N33Q + T231R + N233R + D254R 3.1 +/−0.4 LVA250 N33Q + N94R + T231R + N233R 4.0 +/− 0.1 LVA252 N33Q + D96R +T231R + N233R 2.3 +/− 0.1 LVA254 D27N + N33Q + T231R + N233R 1.5 +/− 0.1LVA256 D27N + N33Q + E56R + T231R + N233R 2.6 +/− 1.2 LVA257 N33Q +L227F + T231R + N233R 2.3 +/− 0.7 LVA272 N33Q + N73Y + G225P + T231R +N233R 2.3 +/− 0.2 LVA273 N33Q + G225P + T231R + N233R 1.9 +/− 0.2 LVA275N33Q + T231R + N233R + D254S 6.2 +/− 1.5 LVA277 N33Q + D96G + T231R +N233R 1.8 +/− 0.1 LVA279 N33Q + D96N + T231R + N233R + D254S 6.1 +/− 0.6LVA280 N33Q + T231R + N233R + D254G 2.1 +/− 0.1 LVA281 N33Q + D130H +T231R + N233R 1.2 +/− 0.0 LVA284 N33Q + E87A + T231R + N233R 2.1 +/− 0.1LVA287 N33Q + T231R + N233R + E239D 1.2 LVA307 N33Q + D111A + T231R +N233R + D254G 2.2 LVA308 N33Q + E210V + T231R + N233R + D254S 5.9 LVA310N11R + N33Q + E210V + T231R + N233R + 7.6 D254S LVA315 N33Q + G91T +G163K + T231R + N233R + 3.4 D254G LVA317 N33Q + G91T + G163K + T231R +N233R + 5.5 D254S LVA319 N11R + N33Q + G91T + G163K + T231R + 6.6N233R + D254S LVA325 Q4R + D27R + N33Q + G91T + N94S + 1.2 D111A +S216P + L227G + T231R + N233R + P256T LVA327 N33Q + G91T + N94S +D111A + V176I + 9.2 T231R + N233R LVA330 Q4R + D27R + N33Q + G91T +N94S + 1.1 D111A + E210D + S216P + L227G + T231R + N233R + P256T LVA331Q4R + D27Q + N33Q + G91T + N94S + 1.2 D111A + S216P + L227G + T231R +N233R + P256T LVA333 N33Q + G91T + N94S + D111A + T231R + 3.5 N233R +P256T LVA334 N33Q + G177A + T231R + N233R 1.0 LVA338 N33Q + T231R +N233R + G246A 1.1 LVA341 D27N + N33Q + G91T + G163K + T231R + 4.2N233R + D254S LVA345 D27Q + N33Q + G91T + G163K + E219D + 2.3 T231R +N233R LVA347 N33Q + G91T + E219D + T231R + N233R 1.2 Diet II (pH 3 ingastric step) LVA055 N33Q + E219D + T231R + N233R 5.1 LVA060 N33Q +W117L + T231R + N233R 2.4 LVA061 D27Q + N33Q + T231R + N233R 2.6 LVA063N33Q + G91T + T231R + N233R 5.1 LVA089 D27S + N33Q + G91A + D96E +L97Q + 2.2 D111A + S216P + T231R + N233R + P256T LVA094 D27R + N33Q +G91N + N94R + D111A + 1.0 T231R + N233R + P256T LVA099 D27R + N33Q +G91T + N94S + D111A + 2.5 S216P + L227G + T231R + N233R + P256T LVA103Q4R + N33Q + T231R + N233R 1.3 LVA113 N33Q + T231R + N233R + Q249R 1.3LVA120 N33Q + D96W + T231R + N233R 1.1 LVA179 N33Q + G91N + T231R +N233R 1.7 +/− 0.6

TABLE 8a Lipase variants with an improved performance in vitro(purified) Improvement Factor (IF) Enzyme tested AST A₂₈₀ Diet I (pH 3in gastric step) LVAR0003 5.2 +/− 0.3 4.6 +/− 0.2 LVAR0013 5.5 +/− 0.74.8 +/− 0.6 LVAR0032 3.7 +/− 1.4 3.3 +/− 1.0 LVAR0050 3.9 +/− 0.4 1.5+/− 0.1 LVAR0058 3.6 +/− 0.4 2.7 +/− 0.4 LVAR0069 5.0 +/− 0.8 1.4 +/−0.2

TABLE 8b Additional lipase variants with an improved performance invitro Lipase Substitutions as compared to SEQ ID NO: 2 IF (AST) Diet I(pH 5 in gastric step) LVA162 N33Q + D167E + T231R + N233R 1.4 +/− 0.2LVA214 N33Q + E87A + T231R + N233R 2.7 +/− 0.4 LVA217 N33Q + E210V +T231R + N233R 2.8 +/− 0.9 LVA218 N33Q + E56K + T231R + N233R 2.4 +/− 0.5LVA220 N33Q + T231R + N233R + D254G 2.0 +/− 0.2 LVA221 N33Q + D96S +T231R + N233R 2.1 +/− 0.4 LVA222 N33Q + D122N + T231R + N233R 1.6 +/−0.2 LVA228 N26A + N33Q + T231R + N233R 1.2 +/− 0.2 LVA229 N33Q + N162T +T231R + N233R 3.5 +/− 1.9 LVA230 N33Q + A150V + N162G + T231R + N233R1.6 +/− 0.3 LVA234 N33Q + T231R + N233R + G240L 2.8 +/− 0.2 LVA308N33Q + E210V + T231R + N233R + D254S 2.9 +/− 0.1 LVA310 N11R + N33Q +E210V + T231R + N233R + 3.0 +/− 0.4 D254S LVA327 N33Q + G91T + N94S +D111A + V176I + 2.0 +/− 0.6 T231R + N233R LVA330 Q4R + D27R + N33Q +G91T + N94S + 1.1 +/− 0.0 D111A + E210D + S216P + L227G + T231R +N233R + P256T LVA347 N33Q + G91T + E219D + T231R + N233R 1.7 +/− 0.3LVA353 N33Q + G163R + T231R + N233R 1.5 +/− 0.1 LVA355 N33Q + G163N +T231R + N233R 2.2 +/− 0.4 LVA357 N33Q + G163C + T231R + N233R 1.6 +/−0.3 LVA359 N33Q + G163Q + T231R + N233R 1.6 +/− 0.0 LVA360 N33Q +G163E + T231R + N233R 1.7 +/− 0.1 LVA362 N33Q + G163H + T231R + N233R1.3 +/− 0.1 LVA364 N33Q + G163I + T231R + N233R 1.5 +/− 0.4 LVA371N33Q + G91K + T231R + N233R 2.1 +/− 0.9 LVA373 N33Q + G91M + T231R +N233R 2.0 +/− 0.4 LVA375 N33Q + G91F + T231R + N233R 1.9 +/− 0.1 LVA379N33Q + G91S + T231R + N233R 1.4 +/− 0.1 LVA381 N33Q + G91W + T231R +N233R 1.5 +/− 0.2 LVA383 N33Q + G91Y + T231R + N233R 1.6 +/− 0.3 LVA391N33Q + G163Y + T231R + N233R 1.9 +/− 0.1 LVA393 N33Q + G163V + T231R +N233R 1.6 +/− 0.5 LVA399 N33Q + G91C + T231R + N233R 1.2 +/− 0.0 LVA411N33Q + G91Y + Q126L + T231R + N233R 2.2 +/− 0.0 LVA412 N33Q + G91M +G161E + T231R + N233R 2.6 +/− 0.2 LVA413 N33Q + V128A + T231R + N233R1.6 +/− 0.1 LVA414 N33Q + V128A + T231R + N233R 1.4 +/− 0.0 LVA415N33Q + G163E + T231R + N233R 1.9 +/− 0.5 LVA416 N33Q + G163V + L185M +T231R + N233R 1.4 +/− 0.3 LVA417 N33Q + G38A + T231R + N233R 1.6 +/− 0.1LVA420 N33Q + G163A + T231R + N233R 1.2 +/− 0.0 LVA421 N33Q + G91T +N94S + D111A + T231R + 1.2 +/− 0.1 N233R LVA438 N33Q + G163M + T231R +N233R 2.4 +/− 0.2 LVA440 N33Q + G91V + T231R + N233R 1.5 +/− 0.2 LVA442N33Q + D111A + T231R + N233R + Q249R 2.6 +/− 0.4 LVA450 D27R + N33Q +G91A + D96E + L97Q + 2.7 +/− 0.1 D111A + T231R + N233R + D254G + P256TLVA451 N33Q + G91T + N94R + T231R + N233R + 3.2 +/− 0.1 D254S LVA453N33Q + G91T + N94R + D111A + W117L + 2.0 +/− 0.5 T231R + N233R LVA454N33Q + W117L + T231R + N233R + D254S 2.3 +/− 0.3 LVA456 N33Q + T231R +N233R + P256T 1.7 +/− 0.1 LVA458 N33Q + T231R + N233R + D242E 2.7 +/−0.0 LVA460 N33Q + E87R + T231R + N233R 3.8 +/− 0.1 LVA461 N33Q + E56R +T231R + N233R 2.4 +/− 0.2 LVA463 N33Q + N162G + T231R + N233R 2.3 +/−0.2 LVA464 N33Q + G91L + T231R + N233R 1.7 +/− 0.1 LVA468 N33Q + E87H +T231R + N233R 2.1 +/− 0.2 LVA470 N33Q + D96N + T231R + N233R + Q249R 2.9+/− 1.2 LVA471 N33Q + G91T + N94R + T231R + N233R + 4.2 +/− 2.7 D254SLVA472 N33Q + L227F + T231R + N233R + D254S 2.0 +/− 0.1 LVA474 N33Q +G163A + T231R + N233R 2.5 +/− 1.1 LVA480 D27R + N33Q + G91T + D96E +D111A + 1.7 +/− 0.6 T231R + N233R + D254S + P256T LVA482 N33Q + G91T +N94R + T231R + N233R 1.9 +/− 0.0 LVA483 N33Q + T231R + N233R + D254A 1.5+/− 0.1 LVA484 N33Q + T231R + N233R + D254N 1.1 +/− 0.0 LVA490 N33Q +T231R + N233R + D254L 2.4 +/− 0.1 LVA492 N33Q + T231R + N233R + D254K3.1 +/− 0.2 LVA494 N33Q + T231R + N233R + D254M 1.8 +/− 0.2 LVA505D27V + N33Q + V60S + G91T + D96W + 4.5 +/− 0.6 T231R + N233R + Q249RLVA506 N33Q + D96N + L227G + T231R + N233R + 2.6 +/− 0.1 Q249R LVA507D27R + N33Q + L227G + T231R + N233R 1.5 +/− 0.2 LVA509 D27R + N33Q +L227G + T231R + N233R + 1.4 +/− 0.1 Q249R LVA512 N33Q + E219D + L227G +T231R + N233R + 3.9 +/− 0.3 Q249R LVA513 D27Q + N33Q + L227G + T231R +N233R + 1.3 +/− 0.0 Q249R LVA516 N33Q + W117L + L227G + T231R + N233R +1.5 +/− 0.1 Q249R LVA518 D5E + N33Q + W117L + L227G + T231R + 1.2 +/−0.2 N233R + Q249R LVA519 D27Q + N33Q + E219D + L227G + T231R + 2.5 +/−0.2 N233R + Q249R LVA520 N33Q + D96E + E219D + L227G + T231R + 3.6 +/−0.0 N233R + Q249R LVA523 D27R + N33Q + E56K + G91N + N94R + 2.0 +/− 0.2D111A + S216P + L227G + T231R + N233R + P256T LVA526 D27R + N33Q +E56Q + D57N + G91N + 2.2 +/− 0.4 N94R + D111 A + S216P + L227G + T231R +N233R + P256T LVA527 D27R + N33Q + E56Q + D57N + G91N + 1.9 +/− 0.7N94R + D111 + S216P + L227G + T231R + N233R + D254S + P256T LVA530D27R + N33Q + E56S + G91N + N94R + 1.5 +/− 0.4 D111A + S216P + L227G +T231R + N233R + P256T LVA532 D27R + N33Q + G91N + N94R + D111A + 1.8 +/−0.1 S216P + L227G + T231R + N233R + D254S + P256T LVA535 D27R + N33Q +G91N + N94R + D111A + 1.8 +/− 0.0 S216P + L227G + T231R + N233R +D254S + P256T LVA540 D27R + N33Q + G91N + N94R + D111S + 1.6 +/− 0.3A155V + S216P + L227G + T231R + N233R + D254S + P256T LVA542 D27R +N33Q + G91N + N94R + D111S + 1.7 +/− 0.2 S216P + L227G + T231R + N233R +D254S + P256T LVA547 N33Q + D111A + T231R + N233R + D254S 2.1 +/− 0.1LVA548 N33Q + D111A + W117L + T231R + N233R + 1.7 +/− 0.4 D254S LVA555N33Q + T231R + N233R + P256N 2.1 +/− 0.1 LVA561 N33Q + T231R + N233R +P256G 1.6 +/− 0.0 LVA562 N33Q + T231R + N233R + P256H 1.2 +/− 0.1 LVA565N33Q + T231R + N233R + P256M 3.0 +/− 0.3 LVA567 N33Q + T231R + N233R +P256W 2.2 +/− 0.4 LVA569 N33Q + T231R + N233R + P256Y 1.7 +/− 0.4 LVA576N33Q + T231R + N233R + P256F 2.0 +/− 1.0 LVA578 N33Q + T231R + N233R +P256V 1.9 +/− 0.5 LVA580 N33Q + G91M + G163W + T231R + N233R 1.4 +/− 0.3LVA581 N33Q + G91M + G163T + T231R + N233R 1.7 +/− 0.6 LVA582 N33Q +G91M + G163D + T231R + N233R 1.7 +/− 0.7 LVA583 N33Q + G91K + G163W +T231R + N233R 1.3 +/− 0.2 LVA586 N33Q + G91T + G163W + T231R + N233R 1.3+/− 0.3 LVA602 N33Q + V176N + T231R + N233R 1.7 +/− 0.7 LVA604 N33Q +V176D + T231R + N233R 1.2 +/− 0.2 LVA614 N33Q + W117F + T231R + N233R1.6 +/− 0.1 LVA622 N33Q + V176I + T231R + N233R 1.3 +/− 0.0 LVA623N33Q + D111N + T231R + N233R 1.1 +/− 0.0 LVA627 N33Q + D111N + G225P +T231R + N233R 1.9 +/− 0.3 LVA629 N33Q + D111N + S216P + T231R + N233R1.4 +/− 0.1 LVA631 D27R + N33Q + G91T + N94R + D111A + 2.4 +/− 0.4S216P + L227G + T231R + N233R LVA632 N33Q + G91M + G163P + T231R + N233R1.5 +/− 0.1 LVA634 N33Q + G91T + G163A + T231R + N233R 1.3 +/− 0.1LVA639 N33Q + W117D + T231R + N233R 1.4 +/− 0.2 LVA640 N33Q + W117H +T231R + N233R 1.3 +/− 0.1 LVA649 N33Q + W117C + T231R + N233R 1.6 +/−0.1 LVA650 N33Q + W117C + T231R + N233R 1.7 +/− 0.1 LVA651 N33Q +W117K + T231R + N233R 1.7 +/− 0.1 LVA653 N33Q + W117V + T231R + N233R1.2 +/− 0.1 LVA656 N11S + N33Q + T231R + N233R 2.0 +/− 0.4 LVA658 N33Q +W117E + V176K + T231R + N233R 2.2 +/− 0.8 LVA659 N33Q + W117G + T231R +N233R 1.8 +/− 0.3 LVA664 N33Q + W117P + T231R + N233R 2.3 +/− 0.7 LVA665N33Q + W117S + T231R + N233R 1.7 +/− 0.1 LVA666 N33Q + W117T + T231R +N233R 1.8 +/− 0.1 LVA667 N33Q + W117I + T231R + N233R 1.3 +/− 0.1 LVA670D27R + N33Q + L227G + T231R + N233R + 4.1 +/− 0.8 Q249R + D254S LVA696N33Q + V176M + T231R + N233R 1.3 +/− 0.2 LVA697 N33Q + V176H + T231R +N233R 1.2 +/− 0.2 LVA700 N33Q + V176A + T231R + N233R 1.2 +/− 0.0 LVA702D27V + N33Q + L227F + T231R + N233R + 1.3 +/− 0.3 Q249R LVA705 N33Q +W117Y + T231R + N233R 1.1 +/− 0.1 LVA707 N33Q + W117Y + V176D + T231R +N233R 1.4 +/− 0.3 LVA715 D27R + N33Q + P136H + L227G + T231R + 1.3 +/−0.2 N233R + Q249R + D254S LVA718 N11R + N33Q + T231R + N233R + T244S 2.0+/− 0.5 LVA721 N33Q + G91T + D96N + D111A + V176I + 3.0 +/− 0.3 T231R +N233R + D254S LVA722 N33Q + G91T + N94S + D111A + V176I + 2.3 +/− 0.2T231R + N233R + D254S LVA723 N33Q + G161A + T231R + N233R 1.5 +/− 0.2LVA731 N33Q + G38I + G177A + T231R + N233R 2.9 +/− 0.6 LVA732 N33Q +N101Q + T231R + N233R 2.5 +/− 0.6 LVA733 N33Q + N94Q + T231R + N233R 2.0+/− 0.1 LVA736 N11Q + N33Q + T231R + N233R 2.0 +/− 0.2 LVA738 N8Q +N33Q + T231R + N233R 1.6 +/− 0.2 LVA740 N33Q + T231R + N233R + N251Q 1.3+/− 0.1 LVA743 N33Q + N200Q + T231R + N233R 1.4 +/− 0.0 LVA744 N33Q +G177A + T231R + N233R 2.3 +/− 0.3 LVA746 N33Q + N73Q + T231R + N233R 1.8+/− 0.2 LVA749 N33Q + I86L + T231R + N233R 1.6 +/− 0.4 LVA753 N33Q +K98I + G163K + T231R + N233R 1.5 +/− 0.3 LVA754 D27R + N33Q + G91T +D96E + D111A + 2.0 +/− 0.4 G163K + T231R + N233R + D254S + P256T LVA755D27R + N33Q + G91T + D96E + D111A + 3.9 +/− 0.3 G163A + T231R + N233R +D254S + P256T LVA770 D27R + N33Q + S216P + L227G + T231R + 1.6 +/− 0.2N233R + Q249R LVA771 N33Q + K98I + G163K + N200Q + T231R + 1.7 +/− 0.5N233R + N251S LVA772 N33Q + G38S + G163K + T231R + N233R 1.7 +/− 0.6LVA774 N33Q G38Y T231R N233R 1.3 +/− 0.2 LVA777 D27R + N33Q + G91T +N94R + D111A + 1.4 +/− 0.2 S216P + L227G + T231R + N233R + P256T LVA778D27R + N33Q + G91T + N94R + D111A + 1.3 +/− 0.0 S216P + L227G + T231R +N233R + P256T LVA782 N33Q + G38N + N73Q + T231R + N233R 1.9 +/− 0.9LVA783 N33Q + G38D + R84E + T231R + N233R 1.8 +/− 0.6 LVA784 N33Q +G38Q + T231R + N233R 1.8 +/− 0.5 LVA786 N33Q + G38I + T231R + N233R 2.2+/− 0.3 LVA788 N33Q + G38K + T231R + N233R 1.4 +/− 0.1 LVA792 N33Q +G38F + T231R + N233R 2.4 +/− 0.2 LVA799 N33Q + G38H + N200Q + T231R +N233R + 2.2 +/− 0.2 N251S LVA800 N33Q + G38L + T231R + N233R 1.8 +/− 0.3LVA804 N33Q + G38P + T231R + N233R 1.5 +/− 0.3 LVA805 N33Q + G38T +T231R + N233R 1.9 +/− 0.1 LVA806 N11R + N33Q + G91T + W117I + G163K +2.6 +/− 0.4 T231R + N233R + D254S LVA808 D27R + N33Q + G38A + G91T +D96E + 3.7 +/− 0.2 D111A + G163K + T231R + N233R + D254S + P256T LVA809N11R + N33Q + G91T + W117I + G163K + 2.6 +/− 0.2 T231R + N233R + D254SLVA811 D27R + N33Q + G38A + G91T + D96E + 2.2 +/− 0.3 D111A + G163A +T231R + N233R + D254S + P256T LVA813 D27R + N33Q + V176Q + L227G +T231R + 2.6 +/− 0.4 N233R + Q249R + D254S LVA814 N33Q + W117I + V176Q +T231R + N233R + 1.8 +/− 0.5 P256A LVA816 N33Q + G38A + G163A + T231R +N233R + 3.1 +/− 0.1 P256A LVA817 N33Q + W117I + V176Q + T231R + N233R2.8 +/− 0.8 LVA818 N33Q + G177A + T231R + N233R + G246A 2.3 +/− 0.8LVA819 E1N N33Q T231R N233R 2.3 +/− 1.2 LVA821 N33Q G38H T231R N233R 2.0+/− 0.4 LVA830 N11R + N33Q + G91T + G163K + V176Q + 1.8 +/− 0.3 T231R +N233R + D254S LVA831 N33Q + K98I + T231R + N233R 1.2 +/− 0.1 LVA834D27R + N33Q + W117I + V176Q + L227G + 1.4 +/− 0.0 T231R + N233R +Q249R + D254S LVA835 N11R + N33Q + G38A + G91T + G163K + 1.9 +/− 0.4T231R + N233R + D254S LVA839 N33Q + G163W + T231R + N233R 1.9 +/− 0.3LVA841 N33Q + G38A + G163A + T231R + N233R 1.9 +/− 0.2 LVA842 D27R +N33Q + G91T + D96E + L97Q + 2.5 +/− 0.9 D111A + T231R + N233R + D254S +P256T LVA844 N33Q + T231R + N233R + D254Q 1.5 +/− 0.2 LVA846 N11R +N33Q + G91T + S115L + G163K + 1.5 +/− 0.2 T231R + N233R + D254S LVA847N11R + N33Q + G91T + G163K + V176W + 1.4 +/− 0.3 T231R + N233R + D254SLVA848 N33Q + G163D + T231R + N233R 1.3 +/− 0.1 LVA849 N33Q + G163D +T231R + N233R 1.2 +/− 0.2 LVA850 N33Q + G163P + T231R + N233R 1.2 +/−0.2 LVA853 E1D + N33Q + G91T + N94R + D111A + 1.2 +/− 0.0 W117L +T231R + N233R + D254S LVA857 N33Q + G91T + N94R + D111A + W117L + 3.8+/− 0.7 V176W + T231R + N233R LVA860 Q4P + D27R + N33Q + G91N + N94R +2.6 +/− 0.4 D111A + L206F + S216P + L227G + T231R + N233R + P256T LVA862D27R + N33Q + T37K + N71I + G91N + 1.7 +/− 0.5 N94R + K98I + D111A +S216P + L227G + T231R + N233R + P256T LVA863 D27R + N33Q + E43K + K46M +I90V + 2.1 +/− 0.1 G91N + N94R + D111A + T114I + S216P + L227G + T231R +N233R + P256T LVA865 N33Q + W117S + T231R + N233R 2.0 +/− 0.2 LVA866N33Q + G61R + V63R + G156R + V176W + 2.1 +/− 0.4 T231R + N233R + P256ILVA869 N33Q + D96N + G156R + V176W + T231R + 2.6 +/− 0.3 N233R LVA871N33Q + G156R + V176W + T231R + N233R + 1.6 +/− 0.3 Q249R LVA873 N33Q +G91T + N94S + D111A + G163T + 1.4 +/− 0.3 V176W + T231R + N233R LVA875N33Q + G91T + N94S + D111A + S115L + 1.6 +/− 0.1 G163T + V176I + T231R +N233R LVA877 N11R + D27R + N33Q + E56Q + D57N + 1.6 +/− 0.2 G91N +N94R + D111S + G163T + S216P + L227G + T231R + N233R + D254S + P256TLVA878 D27R + N33Q + E56Q + D57N + G91N + 1.2 +/− 0.1 N94R + D111S +G163T + S216P + L227G + T231R + N233R + D254S + P256T LVA880 N11R +D27R + N33Q + E56Q + D57N + 2.7 +/− 0.3 G91N + N94R + D111S + S216P +L227G + T231R + N233R + D254S + P256T LVA882 D27R + N33Q + E56Q + D57N +G91N + 2.6 +/− 0.1 N94R + D111S + S216P + L227G + T231R + N233R +D242E + D254S + P256T LVA883 D27R + N33Q + G38A + E56Q + D57N + 3.4 +/−1.1 G91N + N94R + D111S + S216P + L227G + T231R + N233R + D254S + P256TLVA888 Q4R + D27Q + N33Q + G91T + N94S + 1.2 +/− 0.2 E99D + D111A +E210D + S216P + L227G + T231R + N233R + P256L LVA890 N33Q + G38A +G91T + G163A + T231R + 1.6 +/− 0.5 N233R + D254S LVA892 N33Q + G38A +G163A + T231R + N233R + 1.2 +/− 0.2 D254I LVA896 N11R + N33Q + I90L +G163L + T231R + 4.0 +/− 0.4 N233R LVA897 N11R + N33Q + I90L + G163L +T231R + 1.9 +/− 0.5 N233R + D254S LVA899 N11R + N33Q + E56Q + G91T +G163K + 1.3 +/− 0.2 V176Q + T231R + N233R + D254S LVA904 N11R + D27R +N33Q + G91T + D96E + 4.5 +/− 1.6 D111A + G163K + T231R + N233R + D254S +P256T LVA906 N11R + N33Q + G38A + G91T + G112A + 3.6 +/− 0.4 G163A +T231R + N233R + D254S LVA907 N11R + N33Q + G91T + G163K + E210D + 2.2+/− 0.3 T231R + N233R + D254S LVA913 N11R + N33Q + G91T + G163K +T231R + 3.5 +/− 0.9 N233R + D254I LVA915 N11R + N33Q + G91T + G163K +V176T + 1.8 +/− 0.5 T231R + N233R + D254S LVA917 N11R + N33Q + G91T +G163P + T231R + 2.0 +/− 0.1 N233R + D254S LVA919 N11R + N33Q + G91M +G163T + T231R + 1.4 +/− 0.2 N233R + D254S LVA921 N11R + N33Q + G38A +G91T + G163K + 3.9 +/− 1.8 V176D + T231R + N233R + D254S LVA925 N33Q +E56Q + G156R + V176W + T231R + 1.4 +/− 0.2 N233R LVA927 E1D + N33Q +G38A + G91T + N94R + 1.8 +/− 0.2 D111A + W117L + V176W + T231R + N233RLVA928 N33Q + G163K + G177A + T231R + N233R + 3.4 +/− 0.7 G246A LVA929N11R + N33Q + E56Q + G91T + G163K + 2.1 +/− 0.2 T231R + N233R + D254SLVA930 N11R + N33Q + I90L + G163K + T231R + 2.0 +/− 0.2 N233R + D254SLVA933 D27R + N33Q + E56Q + D57N + G91N + 2.7 +/− 0.7 N94R + D111S +S216P + L227G + T231R + N233R + Q249R + D254S + P256T LVA934 D27R +N33Q + E56Q + D57N + G91N + 2.0 +/− 0.2 N94R + D111S + S216P + E219D +L227G + T231R + N233R + D254S + P256T LVA941 N11R + N33Q + I90L + G91T +N94S + 1.5 +/− 0.2 D96E + G163K + T231R + N233R + D254S LVA942 N11R +N33Q + G91T + G163K + V176I + 1.9 +/− 0.1 T231R + N233R + D254S LVA943N11R + N33Q + G91T + G163K + V176Q + 2.1 +/− 0.0 T231R + N233R + D254SLVA944 N11R + N33Q + G91T + G163A + V176T + 1.7 +/− 0.1 T231R + N233R +D254S LVA945 N11R + N33Q + G91T + G163L + V176I + 2.0 +/− 0.0 T231R +N233R + D254S LVA946 N11R + N33Q + G91T + G163L + V176T + 2.2 +/− 0.6T231R + N233R + D254S LVA947 N11R + N33Q + G91T + G163L + T231R + 1.8+/− 0.2 N233R + D254S LVA948 N11R + N33Q + G91T + G163P + T231R + 1.5+/− 0.1 N233R + D254S LVA949 N11R + N33Q + G91T + G163P + V176I + 2.4+/− 0.4 T231R + N233R + D254S LVA950 N11R + N33Q + G91T + G163L +T231R + 2.6 +/− 0.6 N233R + D254S + P256N LVA952 D27R + N33Q + E56Q +D57N + G91N + 1.9 +/− 0.0 N94R + D111S + G163T + S216P + L227G + T231R +N233R + Q249R + D254S + P256T LVA953 Q4R + D27Q + N33Q + G91T + N94S +2.7 +/− 0.4 E99D + D111A + G163A + E210V + S216P + L227G + T231R +N233R + P256L LVA954 Q4R + D27Q + N33Q + G91T + N94S + 3.4 +/− 0.4E99D + D111A + V176I + E210V + S216P + L227G + T231R + N233R + P256LLVA959 N33Q + E210Y + T231R + N233R + D254Y + 1.4 +/− 0.2 I255F LVA961N33Q + L93F + D102Y + T231R + N233R 1.5 +/− 0.1 LVA962 D27R + N33Q +L227G + T231R + N233R + 1.6 +/− 0.1 Q249R + D254S LVA964 N11S + N33Q +T231R + N233R 4.0 +/− 2.7 LVA966 N11R + N33Q + T231R + N233R 2.6 +/− 1.0LVA968 N33Q + G38A + G91T + G163K + T231R + 2.7 +/− 0.7 N233R + D254SLVA969 N33Q + W117Y + V176T + T231R + N233R 2.1 +/− 0.3 LVA970 N8L +N11R + N33Q + G91T + G163K + 3.7 +/− 1.8 T231R + N233R + D254S LVA972E1N + N33Q + G38A + G91T + G163P + 1.8 +/− 0.4 V176F + T231R + N233RLVA973 N11R + N33Q + G38A + G91T + G163P + 2.1 +/− 0.4 V176G + T231R +N233R + D254S LVA976 N11R + N33Q + G91T + G163K + T231R + 3.3 +/− 0.7N233R + D254A + P256F LVA977 N11R + N33Q + G91T + G163K + T231R + 1.6+/− 0.3 N233R + P256F LVA978 N11R + N33Q + G91T + G163K + T231R + 3.0+/− 0.6 N233R + D254S + P256F LVA979 N11R + N33Q + G38A + G91T + G156R +1.6 +/− 0.2 G163K + V176T + T231R + N233R + D254S LVA980 N33Q + G91K +D96S + G163T + T231R + 2.7 +/− 1.4 N233R + Q249R LVA981 N11R + N33Q +G91T + G163N + T231R + 1.9 +/− 0.3 N233R + D254S LVA983 N11R + N33Q +G91T + G163T + T231R + 3.4 +/− 0.6 N233R + D254S LVA984 N11R + N33Q +G91T + G163W + T231R + 5.9 +/− 1.7 N233R + D254S LVA985 N11R + N33Q +G91K + G163K + T231R + 2.2 +/− 0.2 N233R + D254S LVA987 N11R + G23E +N33Q + G91T + G163K + 1.4 +/− 0.0 T231R + N233R + D254S LVA988 N11R +N33Q + G91T + V141E + G163K + 2.0 +/− 0.4 T231R + N233R + D254S LVA989N11R + N33Q + L52R + G91T + G163K + 4.2 +/− 1.2 T231R + N233R + D254SLVA990 N11R + N33Q + G91T + V141L + G163K + 1.8 +/− 0.7 T231R + N233R +D254S LVA991 N11R + N33Q + T37K + G91T + G163K + 3.1 +/− 0.7 T231R +N233R + D254S LVA993 N11R + N33Q + A68V + G91T + G163K + 3.0 +/− 0.5T231R + N233R + D254S LVA994 N11R + N33Q + G91T + G163A + V176I + 3.4+/− 1.9 T231R + N233R + D254S LVA995 N11R + N33Q + T37M + G91T + G163P +2.8 +/− 0.2 V176T + T231R + N233R + D254S LVA997 N11R + N33Q + G91T +G163L + T231R + 2.7 +/− 0.8 N233R + D254S LVA998 N11R + N33Q + G91T +G163K + T231R + 2.2 +/− 0.7 N233R + D254S + P256I LVA999 N33Q + G38S +G156R + G163K + V176W + 6.4 +/− 0.2 T231R + N233R LVA1000 N11R + D27R +N33Q + E56Q + D57N + 2.8 +/− 0.8 G91N + N94R + D111S + G163K + S216P +L227G + T231R + N233R + D254S + P256T LVA1002 N11R + N33Q + G38A +G91T + G163P + 1.4 +/− 0.2 V176G + T231R + N233R + D254S LVA1003 N11R +N33Q + G38A + G91T + G163Q + 2.6 +/− 0.7 V176G + T231R + N233R + D254SLVA1004 N11R + N33Q + G38A + G91T + G163T + 3.2 +/− 0.8 V176G + T231R +N233R + D254S LVA1005 N11R + N33Q + G38A + G91T + N94R + 2.3 +/− 0.4G163P + V176G + T231R + N233R + D254S LVA1006 E1* + N11R + N33Q + G38A +G91N + 1.4 +/− 0.1 N94R + G163P + V176G + T231R + N233R + D254S LVA1007E1N + N11R + N33Q + G38A + G91T + 2.3 +/− 0.3 G163P + V176F + T231R +N233R LVA1008 E1N + F10L + N11R + N33Q + G38A + 2.5 +/− 0.1 G91T +G163P + V176F + T231R + N233R LVA1009 E1N + N33Q + G38A + G91T + G163P +2.7 +/− 0.2 V176F + T231R + N233R + D254S LVA1010 E1N + N33Q + G38A +G91T + D111A + 1.9 +/− 0.3 G163P + V176F + T231R + N233R LVA1011 E1N +N33Q + G38A + G91T + G163P + 2.1 +/− 0.3 V176F + L227F + T231R + N233RLVA1012 E1N + N11R + N33Q + G38A + G91T + 2.4 +/− 0.3 D111A + G163P +V176F + T231R + N233R LVA1013 E1N + N33Q + G38A + G91T + G163P + 3.4 +/−1.2 V176F + L227F + T231R + N233R + D254S LVA1014 E1N + N33Q + G38A +G91T + G163P + 2.2 +/− 0.2 V176F + T231R + N233R + D254S + I255A + P256QLVA1015 E1N + N11R + N33Q + G38A + G91T + 1.9 +/− 0.1 D111A + G163P +V176F + T231R + N233R + D254S LVA1017 N33Q + G156R + V176W + T231R +N233R + 2.4 +/− 0.3 P256I LVA1018 N33Q + G91T + N94S + D111A + G156R +1.9 +/− 0.6 G163T + V176W + T231R + N233R LVA1019 N33Q + G91T + N94S +D111A + G156R + 1.3 +/− 0.3 G163T + V176I + T231R + N233R LVA1021 N11R +N33Q + G38A + G91T + D102G + 2.5 +/− 0.3 S115L + G163K + T231R + N233R +D254S + P256T LVA1023 N11R + N33Q + G38A + G91T + S115L + 2.3 +/− 0.1G163K + T231R + N233R + D254S + P256T LVA1027 E1N + N11R + N33Q + G91T +G163A + 3.1 +/− 0.1 T231R + N233R + G246A + D254S LVA1028 N11R + D27R +N33Q + D57G + G91T + 3.7 +/− 1.4 D96E + D111A + G163K + T231R + N233R +D254S + P256T LVA1029 N33Q + D96N + G156R + V176W + T231R + 3.4 +/− 0.0N233R + Q249R LVA1031 N33Q + I86F + L93F + D102Y + E210Y + 1.4 +/− 0.1L227F + T231R + N233R + D254Y + I255F + L269F LVA1032 N33Q + I86F +L93F + D102Y + E210Y + 1.5 +/− 0.2 L227F + T231R + N233R + D254Y + I255FLVA1033 N11C + N33Q + G91T + G163K + T231R + 2.5 +/− 0.1 N233R + D254SLVA1034 N11L + N33Q + G91T + G163K + T231R + 4.3 +/− 0.0 N233R + D254SLVA1035 N11H + N33Q + G91T + G163K + T231R + 2.2 +/− 0.0 N233R + D254SLVA1036 N11D + N33Q + G91T + G163K + T231R + 4.1 +/− 1.2 N233R + D254SLVA1037 N11R + N33Q + G91T + D96W + G163K + 3.4 +/− 0.0 T231R + N233R +D254S LVA1038 D27R + N33Q + G91T + D96E + L97Q + 5.6 +/− 1.8 D111A +G163K + T231R + N233R + D254S + P2561 LVA1040 N11P + N33Q + G91T +G163K + T231R + 3.0 +/− 1.0 N233R + D254S LVA1041 Q4R + D27N + N33Q +G38A + G91T + 2.5 +/− 0.9 N94S + E99D + D111A + V176I + E210V + S216P +L227G + T231R + N233R + P256L LVA1044 N11R + N33Q + E56Q + G163K +T231R + 2.9 +/− 0.5 N233R + D254S LVA1045 N11R + N33Q + G91T + G163A +T231R + 2.0 +/− 0.1 N233R + D254S LVA1046 N11R + N33Q + G91T + G163P +T231R + 2.8 +/− 0.1 N233R + D254S LVA1048 N11R + N33Q + G91T + G163K +L227G + 2.5 +/− 0.3 P229R + T231R + N233R + D254S

TABLE 8c Additional lipase variants with an improved performance invitro Lipase Substitutions as compared to SEQ ID NO: 2 IF (A280) Diet I(pH 3 in gastric step) LVA012 D27R + N33Q + G91A + D96E + L97Q + 2.5 +/−0.4 D111A + T231R + N233R + P256T LVA023 N33Q + E210D + T231R + N233R1.9 +/− 0.2 LVA027 N33Q + T231R + N233R 1.8 +/− 0.2 LVA041 N33Q +D111A + T231R + N233R 2.0 +/− 0.3 LVA043 N33Q + G91T + T231R + N233R 3.1LVA061 D27Q + N33Q + T231R + N233R 1.7 +/− 0.4 LVA139 Q9H + N33Q +D102E + T231R + N233R 2.3 +/− 1.0 LVA216 N33Q + E56Q + T231R + N233R 1.3+/− 0.1 LVA231 N33Q + I90L + G163L + T231R + N233R 1.3 +/− 0.2 LVA238D27R + N33Q + G91A + D96E + D111A + 1.3 +/− 0.3 T231R + N233R + D254G +P256T LVA245 N33Q + N39S + T231R + N233R 1.7 +/− 0.7 LVA250 N33Q +N94R + T231R + N233R 3.3 +/− 1.8 LVA275 N33Q + T231R + N233R + D254S 4.3+/− 2.7 LVA315 N33Q + G91T + G163K + T231R + N233R + 1.4 +/− 0.2 D254GLVA317 N33Q + G91T + G163K + T231R + N233R + 1.8 +/− 0.4 D254S LVA319N11R + N33Q + G91T + G163K + T231R + 2.2 +/− 0.3 N233R + D254S LVA341D27N + N33Q + G91T + G163K + T231R + 2.3 +/− 0.1 N233R + D254S LVA348N33Q + T231R + N233R 1.4 +/− 0.2 LVA349 K98I + T231R + N233R + N251S 1.4+/− 0.2 LVA368 N33Q + G163P + T231R + N233R 1.7 +/− 0.1 LVA370 N33Q +G163D + T231R + N233R 1.3 +/− 0.1 LVA387 N33Q + G163T + T231R + N233R1.1 +/− 0.1 LVA389 N33Q + G163W + T231R + N233R 1.3 +/− 0.0 LVA437N33Q + G38A + G163A + T231R + N233R 1.4 +/− 0.0 LVA444 N33Q + D111A +T231R + N233R + D254S 1.2 +/− 0.0 LVA473 D27R + N33Q + G91T + D96E +L97Q + 1.2 +/− 0.2 D111A + T231R + N233R + D254S + P256T LVA553 N33Q +T231R + N233R + P256A 1.2 +/− 0.0 LVA566 N33Q + T231R + N233R + P256S1.2 +/− 0.1 LVA620 N33Q + G91T + N94S + D111A + V176I + 1.2 +/− 0.1T231R + N233R + D254S LVA672 N33Q + S115L + T231R + N233R 1.1 +/− 0.0LVA675 N33Q + G38A + G91T + G163K + T231R + 1.6 +/− 0.2 N233R + D254SLVA714 D27V + N33Q + G91A + N94R + D111A + 1.1 +/− 0.0 G163K + L227F +T231R + N233R + Q249R + D254S LVA773 D27R + N33Q + G38A + G91T + D96E +1.3 +/− 0.1 D111A + T231R + N233R + D254S + P256T LVA828 N33Q + G91A +N94K + D111A + G163K + 1.6 +/− 0.3 L227F + T231R + N233R + Q249R LVA829N33Q + G91A + N94K + D111A + G163K + 1.7 +/− 0.0 L227F + T231R + N233R +Q249R + D254S LVA955 N33Q + G91T + K98I + T114I + G163K + 3.3 +/− 1.0T231R + N233R + D254S LVA956 N33Q + G91T + K98I + G163K + T231R + 1.8+/− 0.4 N233R + D254S + P256L LVA957 N33Q + G91T + T114I + G163K +T231R + 2.3 +/− 0.5 N233R + D254S + P256L

TABLE 8d Additional lipase variants with an improved performance invitro Lipase Substitutions as compared to SEQ ID NO: 2 IF (A280) Diet I(pH 5 in gastric step) LVA012 D27R + N33Q + G91A + D96E + L97Q + 2.1 +/−0.5 D111A + T231R + N233R + P256T LVA013 D27R + N33Q + G91A + D96E +L97Q + 1.6 +/− 0.0 D111A + T231R + N233R + P256T LVA023 N33Q + E210D +T231R + N233R 3.2 +/− 1.1 LVA041 N33Q + D111A + T231R + N233R 2.2 +/−0.6 LVA043 N33Q + G91T + T231R + N233R 3.9 +/− 1.1 LVA061 D27Q + N33Q +T231R + N233R 2.4 +/− 0.8 LVA139 Q9H + N33Q + D102E + T231R + N233R 1.5+/− 0.3 LVA208 N33Q + G91T + G163K + T231R + N233R 1.4 +/− 0.1 LVA216N33Q + E56Q + T231R + N233R 1.9 +/− 0.2 LVA231 N33Q + I90L + G163L +T231R + N233R 2.0 +/− 0.0 LVA250 N33Q + N94R + T231R + N233R 1.9 +/− 0.5LVA275 N33Q + T231R + N233R + D254S 1.8 +/− 0.5 LVA315 N33Q + G91T +G163K + T231R + N233R + 1.9 +/− 0.3 D254G LVA317 N33Q + G91T + G163K +T231R + N233R + 2.1 +/− 0.4 D254S LVA319 N11R + N33Q + G91T + G163K +T231R + 2.3 +/− 0.8 N233R + D254S LVA341 D27N + N33Q + G91T + G163K +T231R + 2.4 +/− 0.1 N233R + D254S LVA348 N33Q + T231R + N233R 1.6 +/−0.3 LVA349 K98I + T231R + N233R + N251S 1.7 +/− 0.5 LVA368 N33Q +G163P + T231R + N233R 1.7 +/− 0.1 LVA370 N33Q + G163D + T231R + N233R2.0 +/− 0.4 LVA387 N33Q + G163T + T231R + N233R 1.4 +/− 0.0 LVA389N33Q + G163W + T231R + N233R 1.5 +/− 0.0 LVA437 N33Q + G38A + G163A +T231R + N233R 1.9 +/− 0.3 LVA444 N33Q + D111A + T231R + N233R + D254S1.2 +/− 0.2 LVA449 D27R + N33Q + G91A + D96E + L97Q + 1.4 +/− 0.1D111A + T231R + N233R + D254S + P256T LVA473 D27R + N33Q + G91T + D96E +L97Q + D111A + 1.4 +/− 0.4 T231R + N233R + D254S + P256T LVA486 N33Q +T231R + N233R + D254Q 1.9 +/− 0.1 LVA488 N33Q + T231R + N233R + D254I1.6 +/− 0.1 LVA503 N33Q + S216P + L227G + T231R + N233R + 1.3 +/− 0.1Q249R LVA553 N33Q + T231R + N233R + P256A 1.1 +/− 0.1 LVA564 N33Q +T231R + N233R + P256L 1.2 +/− 0.1 LVA566 N33Q + T231R + N233R + P256S1.7 +/− 0.2 LVA620 N33Q + G91T + N94S + D111A + V176I + 1.5 +/− 0.2T231R + N233R + D254S LVA672 N33Q + S115L + T231R + N233R 1.7 +/− 0.1LVA675 N33Q + G38A + G91T + G163K + T231R + 2.2 +/− 0.4 N233R + D254SLVA713 D27V + N33Q + G91A + N94R + D111A + 2.7 +/− 0.2 G163K + L227F +T231R + N233R + Q249R LVA714 D27V + N33Q + G91A + N94R + D111A + 1.2 +/−0.0 G163K + L227F + T231R + N233R + Q249R + D254S LVA734 N33Q + G161A +T231R + N233R 1.3 +/− 0.1 LVA801 N33Q G38M T231R N233R 3.3 +/− 0.9LVA803 N33Q G38F T231R N233R 3.2 +/− 0.4 LVA828 N33Q + G91A + N94K +D111A + G163K + 2.0 +/− 0.9 L227F + T231R + N233R + Q249R LVA829 N33Q +G91A + N94K + D111A + G163K + 2.8 +/− 1.5 L227F + T231R + N233R +Q249R + D254S LVA955 N33Q + G91T + K98I + T114I + G163K + 4.2 +/− 1.7T231R + N233R + D254S LVA956 N33Q + G91T + K98I + G163K + T231R + 2.6+/− 0.8 N233R + D254S + P256L LVA957 N33Q + G91T + T114I + G163K +T231R + 2.1 +/− 0.3 N233R + D254S + P256L

Example 8 Lipase Variants with Improved Stability at pH 3 in thePresence of Pepsin

The variants of Table 6 in Example 5 were screened for stability at pH 3in the presence of pepsin, together with the variants of Table 9 below.

The variants were selected from two randomly mutagenized yeast librariesof amino acids 21 to 100 of SEQ ID NO: 1 and from a targeted yeastlibrary of SEQ ID NO: 1 with the following changes targeted: D27X, E43X,E56X, D57DA, D62DA, E87EK, D96DL, E99X, D111X, D234X Q249QR, D254DN,from a targeted yeast library of SEQ ID NO:1 with G91T and G163K withthe following changes targeted: N11R, D27RQNV, G38X, D96EW, K98X, T114I,K163WA, E210VD, R231I, D254SGQIK and P256TA, from a randomly mutagenizedlibrary of SEQ ID NO: 1 with G91T and G163K, from a randomly mutagenizedlibrary of SEQ ID NO:1 with D27R, G91N, N94R, D111A, S216P, L227G andP256T or were site-directed variants generated of SEQ ID NO: 1. Theyeast is Sachharoymces cerevisiae JG169 (MATα; ura3-52; leu 2-3, 112;his 3-D200; pep 4-113; prd::HIS3; prbl::LEU2; cir+).

TABLE 9 Lipase variants III Variant designation Substitutions ascompared to SEQ ID NO: 2 LVAR0002b T32I + G91V + T231R + N233R LVAR0011aG91A + T231R + N233R LVAR0014 N33Y + G91W + N94K + T231R + N233RLVAR0015 P42L + D57N + G91E + T231R + N233R LVAR0016 K98I + T231R +N233R LVAR0017 V60L + G91V + T231R + N233R LVAR0048 E43K + E56S + E87K +T231R + N233R LVAR0055 E43V + G91R + T231R + N233R LVAR0059 E43M +E87K + D96L + E99P + T231R + N233R LVAR0066 E43D + E56A + D57A + E87K +D111A + T231R + N233R LVAR0068 E87K + L147S + T231R + N233R LVAR0070E43D + E87K + D96L + E99P + E239V + T231R + N233R LVAR0071 E43K + E56A +E87K + D234K + T231R + N233R LVAR230 D27R + N33Q + E43K + K46M + I90V +G91N + N94R + D111A + T114I + S216P + L227G + T231R + N233R + P256TLVAR226 G23E + D27R + N33Q + L52R + G91N + N94R + D111A + T114I +V141E + S216P + L227G + T231R + N233R + P256T LVAR287 N33Q + G38W +G91T + T114I + G163K + E210D + T231R + N233R + P256T LVAR288 D27I +N33Q + G91T + D96E + K98T + T114I + G163K + E210D + T231R + N233R +P256T LVAR280 N33Q + G91T + D96E + K98T + T114I + T231R + N233R + G163SLVAR286 N33Q + G38W + G91T + T114I + G163K + E210V + T231R + N233RLVAR214 Q4P + D27R + N33Q + G91N + N94R + D111A + R205I + L206F +S216P + L227G + T231R + N233R + P256T LVAR281 N33Q + G91T + D96E +K98T + T114I + G163K + E210D + T231R + N233R LVAR205 D27R + N33Q +T37K + N71I + G91N + N94R + K98I + D111A + S216P + L227G + T231R +N233R + P256T LVAR215 Q4H + D27R + N33Q + G91N + N94R + D111A + V154L +S216P + L227G + T231R + N233R + P256T LVAR277 N33Q + G91T + D96E +K98T + T114I + G163S + E210V + T231R + N233R + D254K + P256A LVAR282N33Q + G91T + T114I + G163K + E210D + T231R + N233R + D254G + P256ALVAR209 D27R + N33Q + L52I + V60E + G91N + N94R + D111A + T114I +V168M + E210D + S216P + L227G + T231R + N233R + P256T LVAR223 D27R +N33Q + G91N + N94R + D111A + T114I + R179T + S216P + L227G + T231R +N233R + P256T LVAR231 D27R + A30V + N33Q + G91N + N94R + G109A + D111A +G190D + S216P + L227G + T231R + N233R + P256T LVAR204 D27R + N33Q +G91N + N94R + K98I + D111A + N162S + S216P + L227G + T231R + N233R +P256T LVAR235 N26H + D27R + N33Q + G91N + N94R + D111A + V154F + G190C +S216P + L227G + T231R + N233R + P256T LVAR284 D27N + N33Q + G91T +T114I + G163S + E210D + T231R + N233R + P256T LVAR225 D27R + N33Q +G91N + N94R + D111A + S216P + L227G + T231R + N233R LVAR205 D27R +N33Q + T37K + N71I + G91N + N94R + K98I + D111A + S216P + L227G +T231R + N233R + P256T LVAR283 D27R + N33Q + G91T + T114I + G163W +E210D + T231R + N233R LVAR219 D27R + N33Q + G91N + N94R + K98I + D111A +S216P + L227G + T231R + N233R + P256T LVAR220 D27R + N33Q + G91N +N94R + L97M + D111A + S216P + T226N + L227G + T231R + N233R + P256T +L269H LVAR216 D27R + N33Q + G91N + N94R + D111A + V154I + S216P +L227G + T231R + N233R + P256T LVAR290 N33Q + G91T + T114I + E210V +T231R + N233R + D254K + P256A LVAR218 D27R + N33Q + N71S + G91N + N94R +D111A + H135D + S216P + L227G + T231R + N233R + P256T LVAR285 N33Q +G91T + T114I + G163K + E210D + T231R + N233R LVAR208 D27R + N33Q +I76T + G91N + N94R + R108M + D111A + S216P + L227G + T231R + N233R +P256T LVAR207 D27R + N33Q + N39S + G91N + N94R + D111A + S216P + L227G +T231R + N233R + P256T LVAR234 D27R + N33Q + A49T + G91N + N94R + D111A +Y138F + G163R + S216P + L227G + T231R + N233R + P256T LVAR828 N33Q +G91A + N94K + D111A + G163K + L227F + T231R + N233R + Q249R LVAR955N33Q + G91T + K98I + T114I + G163K + T231R + N233R + D254S LVAR956N33Q + G91T + K98I + G163K + T231R + N233R + D254S + P256LPrinciple

The screening process measures residual lipase activity after a 3 hourtreatment at pH 3.0 and room temperature in the presence of 75 ug/mLpepsin. The residual lipase activity is measured in a rate assaymonitoring activity over time to allow very high activity and very lowactivity lipases to be detected during the screening event.

In primary screens of variants, a sufficiently large dilution isperformed on the broth sample in order to minimize the effects of mediaor fermentation components on the test conditions. Variants that make itpast the primary phase undergo more testing in the follow up screeningby adding more dilutions and replicates of the test samples.

Materials and Methods

Primary Screen Medium:

1.7 g of yeast nitrogen base (YNB) with ammonium sulfate (Bio 101, Cat#4027-532), 0.8 g of Complete Supplement Mixture-Uracil (CSM-ura) w/40mg adenine (ADE) (Bio 101, Cat #4512-722), 5 g of Casamino acids (BD,Cat #223050), 100 ml of 50% Glucose, 50 ml of 0.5 M K₂HPO₄ (Potassiumphosphate-dibasic), 1 ml of 100 mM CuSO₄-5H₂O (JD Baker, Cat#1843-01), 1ml of 100 mg/mL ampicillin in a total volume adjusted to 1 l withde-ionized water. The medium was filter sterilized and stored at 4° C.

Optimized Medium:

6.7 g of YNB with ammonium sulfate (Bio 101, Cat #4027-532), 5.9 g ofsuccinic acid (Sigma S-9512), 0.8 g of CSM-ura w/40 mg ADE (Bio 101, Cat#4512-722), 20 g of galactose (Sigma, Cat#G-0625), 10 g of glucose, 1 mlof 100 mM CuSO₄-5H₂O and 1 ml of 100 mg/mL ampicillin. The pH isadjusted to 6.6 with NaOH and the volume is adjusted to 1 l withde-ionized water. The medium is filter sterilized and stored at 4° C.

Seed Culture Medium:

Mix following ingredients: 6.7 g of YNB with ammonium sulfate (Bio 101,Cat #4027-532), 5 g of Casamino acids (BD, Cat #223050), 100 ml of 0.5 Msuccinic acid (Sigma S-9512), 855 ml of de-ionized H₂O. Autoclave themixture. Add 2 ml of 10 mg/mL chloramphenicol and 40 ml of 50% Glucose.Store at 4° C.

Stock Solutions for Making Substrate and Pepsin Treatment:

-   1. 10% Triton-X100 (w/v)-   2. 1 M TRIS, pH 8.0-   3. 10% (680 mM) CaCl₂*2H₂O-   4. 100 mM Citric Acid, pH 3.0-   5. 5 mg/mL Porcine Pepsin (Sigma P-6887, 3280 U/mg solid, 3370 U/mg    protein) made up in 100 mM Citric Acid, 0.01% Triton-X100-   6. 50 mM 4-nitrophenol (PNP) Palmitate made up in 10% Triton-X100    Substrate for Lipase Activity Assay:

1 mM PNP-Palmitate, 1.2% Triton-X100, 4 mM CaCl₂, 100 mM TRIS, pH 8.0

Pepsin Treatment Solution:

150 ug/mL pepsin, 4 mM CaCl₂, 0.01% Triton-X100, 50 mM Citrate, pH 3.0

Diluent:

0.01% Triton-X100, 10 mM NaCl

Enzymes:

Broth samples of lipase variants for primary screens were derived fromindividual clones picked off agar plates into Primary Screen Medium in96-well plates.

Growth of Cultures:

Three media recipes were used to grow lipase variants. Since expressionlevel at primary screen level is not critical, Primary Screen Medium wasused for earlier screen stage. Single lipase variant expressing yeastcolonies were picked Into 180 uL of Primary Screen Medium in 96-wellplates and grown at 30° C. and 250 rpm for 4-6 days for the primaryscreening samples.

For the secondary screening, 20 uL of culture from the primary screeningplate was transferred into 1 mL of Seed Culture Medium in 24-well platesand grown overnight at 30° C. and 250 rpm. Expression of the lipase wasachieved by inoculating 20 uL of the Seed Culture into 1 mL of OptimizedMedium in 24-well plates at 30° C. and 250 rpm for 4-6 days.

For any subsequent screening, single yeast colonies were picked into 1mL of Seed Culture Medium in 24-well plates and grown overnight at 30°C. and 250 rpm. Expression of the lipase was achieved by inoculating 20uL of the Seed Culture into 1 mL of Optimized Medium in 24-well platesat 30° C. and 250 rpm for 4-6 days. Optimized medium was used forgrowing in a 24-well plate and a shake flask to maximize the proteinexpression level.

Screening Procedure:

In primary screens, samples were diluted 25-fold in diluent, then 5 uLwas added to 384 well plates containing 5 uL of either diluent or pepsintreatment solution.

After 3 hours at room temperature, substrate was added to each sample asfollows: The pepsin-treated samples were mixed with 55 uL substrate+5 uLdiluent, the untreated samples were mixed with 55 uL substrate+5 uLpepsin-treatment solution. (The pepsin pH is 3.0 and withoutcompensating for the change in pH, the activity assay will be run at twodifferent pH values, so this normalizes the assay pH without having aneffect on stability of lipase (by adding it at the end when the pH ofthe substrate is sufficient to raise the overall pH to 8.0, where pepsinis not active). OD₄₀₅ readings were taken 6 times per 384-well plate; asearly as 15 minutes after substrate addition and as long as 18 hoursafter substrate addition and were expressed as mOD (milli OD) per hour.Data that falls in the linear range was collected and the residuallipase activity of each pepsin-treated sample was compared with theresidual lipase activity of the corresponding untreated sample. This isreported below as % residual activity (% RA); calculated by dividing therate of the treated condition by the rate of the untreated condition andmultiplying the result by 100.

Automation may be accomplished using a system comprised of a Biomek FXworkstation which functions to move plates and perform pipetting steps,a DXT 880 plate reader to record data from the assay plates, a carouseland conveyor belt system to transport plates to and from theworkstation. This procedure accepts samples in 96-well plate formats,performs dilutions in the same format, then uses 384-well plates for thetreatment and assay steps.

Results:

The relative residual activities for each lipase variant as measuredafter the secondary screen are shown in Table 10 below. Each of thesevariants has an improved RA (stability at pH 3 in the presence ofpepsin) as compared to the lipase of amino acids 1-269 of SEQ ID NO: 2.

TABLE 10 Residual lipase activity after incubation at pH 3 in thepresence of pepsin Enzyme tested Residual Activity (RA), % SEQ ID NO: 22.3 SEQ ID NO: 1 9.7 LVAR0002b 37 LVAR0003 37 LVAR0011a 58 LVAR0013 56LVAR0014 80 LVAR0015 70 LVAR0016 37 LVAR0017 62 LVAR0045 33 LVAR0046 61LVAR0047 45 LVAR0048 39 LVAR0050 45 LVAR0051 40 LVAR0052 58 LVAR0053 44LVAR0054 30 LVAR0055 66 LVAR0056 37 LVAR0057 71 LVAR0058 42 LVAR0059 36LVAR0061 63 LVAR0062 31 LVAR0063 59 LVAR0064 33 LVAR0065 40 LVAR0066 34LVAR0067 32 LVAR0068 57 LVAR0069 45 LVAR0070 52 LVAR0071 60 LVAR0072 52LVAR0101 95 LVAR0102 76 LVAR0106 86 LVAR230 91 LVAR226 100 LVAR287 96LVAR288 94 LVAR280 93 LVAR286 91 LVAR214 90 LVAR281 90 LVAR205 89LVAR215 88 LVAR277 87 LVAR282 87 LVAR209 86 LVAR223 85 LVAR231 84LVAR204 84 LVAR235 83 LVAR284 83 LVAR225 81 LVAR205 80 LVAR283 80LVAR219 80 LVAR220 79 LVAR216 79 LVAR290 78 LVAR218 78 LVAR285 78LVAR208 77 LVAR207 76 LVAR234 75 LVAR828 75 LVAR955 96 LVAR956 85

Example 9 Lipase Variants with Improved Activity in the Presence of BileSalts

In order to identify lipase variants with improved activity in thepresence of bile salts the following assay was developed. The assaymeasures lipase activity in the presence of 2 mM bile salts as comparedto conditions without bile salts. The test is set up such that lipaseactivity is measured in a rate assay monitoring activity over time toallow very high activity and very low activity lipases to be detected.This assay is automated to precisely control the timing of the additionof reagents and adjustments of the pH of the reactions from pH 5.0,where the lipase reacts with the substrate at low pH, to pH 8.0, a pHthat allows the released PNP group to be read at OD 405. The plates areread immediately after the pH adjustment occurs.

Stock Solutions for Assay Substrate and Bile Salts Treatment:

-   1. 10% Triton-X100 (w/v)-   2. 100 mM TRIS, pH 8.0-   3. 100 mM Succinate, pH 5.0-   4. 10% (680 mM) CaCl₂*2H₂O-   5. 20 mM Bile Salts (Sigma B-8756) made up in distilled water-   6. 50 mM 4-nitrophenol (PNP) Oleate made up in 10% Triton-X100    Substrate for Lipase Activity Assay:

1 mM PNP-Oleate, 1.2% Triton-X100, 2 mM CaCl₂, 25 mM Succinate, pH 5.0

Diluent:

0.01% Triton-X100, 10 mM NaCl

Enzymes:

Purified lipase samples are diluted in diluent to approximately 8micrograms/mL for the automated method. The concentration of thepurified lipase samples was determined from the absorbance at 280 nmusing the extinction coefficient 1.24 A₂₈₀/mg.

Liquid Based Bile Salts Assay Screening Procedure:

Enzyme samples are diluted 25-fold and 200-fold in diluent, then 10micro-L is added to either 23 micro-L water or 23 micro-L 20 mM Bilesalts in 96-well plate reaction plates. After this, 200 micro-L of thesubstrate (1 mM PNP Oleate in 25 mM Succinate, 2 mM CaCl₂, 1.2%Triton-X100, pH 5.0), is added and mixed. Immediately after mixing, 60micro-L is removed and 15 micro-L is pipetted into four separate384-well plates where the 4 quadrants (4×96) are used to set up the “+”and “−” bile salts conditions for each of 2 dilutions of the lipasesamples. The four 384-well plates are used to set up 4 time points (suchthat each plate has the 2 dilutions of the lipase each “+” and “−” bilesalts). After 1, 2, 3, and 4 hours 60 micro-L of 100 mM TRIS, pH 8.0 isadded to the appropriate plate and quadrant and read (OD 405 and OD 540)immediately. ODs between ˜0.1 and 0.475 are the linear range used forthis assay. A ratio of the activity in the presence of bile salts at pH5.0 is expressed as a percentage by calculating the average of alllinear data corrected for time and dilution for the “bile salts”activity divided by the average of all linear data corrected for timeand dilution for the “no bile salts” activity. The ratio of activityversus the reference (variant N33Q of SEQ ID NO: 1) is calculated bydividing the ratio of the variants plus and minus bile salts by theratio of the reference plus and minus bile salts and is reported as afold improvement (e.g., 3× means 300%).

TABLE 11 Bile Salt Ratio Improvement Variant vs Reference Substitutionsas compared to SEQ ID NO: 2 LVAR714 3X D27V + N33Q + G91A + N94R +D111A + G163K + L227F + T231R + N233R + Q249R + D254S LVAR828 3X N33Q +G91A + N94K + D111A + G163K + L227F + T231R + N233R + Q249R LVAR1042 4XG23E + D27R + N33Q + L52R + G91N + N94R + D111A + T114I + V141E +S216P + L227G + T231R + N233R + P256T LVAR1043 3X D27R + N33Q + E43K +K46M + I90V + G91N + N94R + D111A + T114I + S216P + L227G + T231R +N233R + P256T LVAR861 3X G23E + D27R + N33Q + L52R + G91N + N94R +D111A + T114I + V141E + S216P + L227G + T231R + N233R + P256T LVAR863  2.5X D27R + N33Q + E43K + K46M + I90V + G91N + N94R + D111A + T114I +S216P + L227G + T231R + N233R + P256T LVAR290 3X N33Q + G91T + T114I +E210V + D254K + P256A LVAR277 2X N33Q + G91T + D96E + K98T + T114I +G163S + E210V + D254K + P256A LVAR209 2X L52I + V60E + T114I + V168M +E210D LVAR234 2X D27R + N33Q + A49T + G91N + N94R + D111A + Y138F +G163R + S216P + L227G + T231R + N233R + P256T LVAR208 2X D27R + N33Q +I76T + G91N + N94R + R108M + D111A + S216P + L227G + T231R + N233R +P256T LVAR230 2X D27R + N33Q + E43K + K46M + I90V + G91N + N94R +D111A + T114I + S216P + L227G + T231R + N233R + P256T

Example 10 In Vivo Digestibility Trial (Screening Test)

Selected purified Humicola lanuginosa lipase variants of the inventionwere studied in a lipase screening test in female Göttingen minipigs(Ellegaard) with induced Pancreatic Exocrine Insufficiency (PEI). Theamino acid sequences of the variants are found in Tables 1, 7 and 8. Theefficacy was compared to that of variant N33Q of the lipase having aminoacids 1-269 of SEQ ID NO: 1 (variant LV2934; reference lipase of Table12). Pancreatic Exocrine Insufficiency (PEI) was induced in the minipigsby ligation of the pancreatic duct, and they were also fitted with anileo-caecal re-entrant cannula, all under isofluorane anaesthesia and ata weight of about 25 kg, as described in Tabeling et al. (Tabeling etal. (1999): “Studies on nutrient digestibilities (pre-caecal and total)in pancreatic duct-ligated pigs and the effects of enzyme substitution”,J. Anim. Physiol. A. Anim. Nutr. 82: 251-263) and in Gregory et al.(Gregory et al. (1999): “Growth and digestion in pancreatic duct ligatedpigs, Effect of enzyme supplementation” in “Biology of the Pancreas inGrowing Animals” (Pierzynowski & Zabielski eds), Elsevier Science BV,Amsterdam, pp 381-393). A period of at least 4 weeks was allowed forrecovery from surgery, before studies were commenced. Prior to studybegin, the PEI status of each pig was confirmed via the stoolchymotrypsin test (commercially available from Immundiagnostik AG,Wiesenstrasse 4, D-64625 Bensheim, Germany, with catalogue No. K 6990).

Assay

The screening test for lipase activity was performed in two groups of 3or 4 PEI minipigs. During the studies, the pigs were housed in modifiedmetabolism cages and allowed free access to water and fed two meals perday.

Test Meal

247.2 g milk; 1×87 g sachet Calshake from Fresenius Kabi (2077KJ/100 g);29.9 g olive oil; 9.88 g Methocel (Methocel E5, from Colorcon GmbH); and0.368 g chromic oxide. Calshake contains 24.4% fat, 3.3% lactose, 64.9%carbohydrate (49% sugar), 4.3% protein.

The milk and chromic oxide were homogenized with an Ultraturaxhomogenizer (9500 rpm, ca. 1 min), after which the oil was mixed in andagain homogenized for 1-2 minutes. Then the Calshake was mixed in(stirred with mixer for 1-2 min) and finally the Methocel was slowlyadded while mixing with the Ultraturax, and the whole meal was thenhomogenized for approximately 3 minutes.

Performance

To assess lipase efficacy, the pigs were fed a single test meal(containing 51.6 g fat) into which differing amounts of a referencelipase or similar amounts of lipase variants were mixed immediatelybefore feeding.

The reference lipase LV2934 was dosed at 0.124, 1.24, 4.96, or 18.61 mgenzyme protein/meal (corresponding to 500, 5000, 20000, and 75000 FIP Ulipase/meal, respectively), and the lipase variants of the inventionwere also dosed according to mg enzyme protein (1.24, 4.96, and 18.61mg/meal), in order to compare the in vivo efficacy with LV2934. Thestudies were performed according to a Latin Square design.

Ileal chyme was collected for a total of 8 h after first appearance ofthe meal marker in the ileum (green chyme) and two hour samples werefrozen at −20° C. At least one day washout was allowed between separatedeterminations. A low-fat, liquid meal was given in the evening beforeeach test to reduce the likelihood of interference of meal contents fromnon-test meals.

Analysis

The frozen ileal chyme samples were freeze-dried, milled and analysedfor dry matter (DM) and fat (Naumann & Bassler 1993; Die chemischeUntersuchung von Futtermitteln, 3. edition, VDLUFA-Verlag, Darmstadt(VDLUFA=Verband Deutscher Landwirtschaftlicher Untersuchungs- undForschungsanstalten). DM was estimated by weight after freeze-dryingfollowed by 8 h incubation at 103° C.; crude fat content of the driedsample was determined by acid hydrolysis and petrol ether extractionusing a filter bag technique in an ANKOMXT15 extractor (which isavailable from Ankom Technology, Macedon, N.Y., US; capable ofperforming 15 extractions at a time); Cr₂O₃ was oxidized to chromate andchromium content was calculated as described by Petry and Rapp inZeitung für Tierphysiologie (1970), vol. 27, p. 181-189 (Petry & Rapp,1970, Z. Tierphysiol. 27: 181-189) via extinction at 365 nm(spectrophotometer).

Digestibility values (coefficient of fat absorption; CFA) were estimatedby the marker method according to the formula:

${C\; F\; A\mspace{14mu}(\%)} = {100 - {\frac{\left\lbrack {\%\mspace{14mu}{Cr}_{2}O_{3}\mspace{14mu}{in}\mspace{14mu}{feed}} \right\rbrack \cdot \left\lbrack {\%\mspace{14mu}{fat}\mspace{14mu}{in}\mspace{14mu}{ileal}\mspace{14mu}{chyme}} \right\rbrack}{\left\lbrack {\%\mspace{14mu}{Cr}_{2}O_{3}\mspace{14mu}{in}\mspace{14mu}{ileal}\mspace{14mu}{chyme}} \right\rbrack \cdot \left\lbrack {\%\mspace{14mu}{fat}\mspace{14mu}{in}\mspace{14mu}{feed}} \right\rbrack} \cdot 100}}$Results and Conclusion

The CFA results are shown in Table 12. The lipase dosage is indicated inmilligram of enzyme protein per meal (mg/meal).

TABLE 12 Effect of lipase variants of the invention on CFA (Coefficientof Fat Absorption) Lipase 0.124 1.24 4.96 18.61 variant 0 (mg/meal)****(mg/meal)**** (mg/meal)**** (mg/meal)**** No lipase 14.56 +/− 5.94Control 85.8 +/− 3.2 animals (not PEI) Reference 17.53 +/− 4.95*** 35.41+/− 5.41* 54.69 +/− 1.52** 65.48 +/− 5.88* lipase (LV2934)**** LVA12930.02 58.47 75.47 LVA147 41.44 54.84 74.00 LVA238 37.74 57.33 72.05LVA315 37.03 64.87 80.91 LVA317 43.7  68.57 81.14 LVA319 27.07 63.7273.43 83.82 LVA368 34.21 51.45 75.50 *Standard Deviation calculated from6 independent tests and including the testing of LVA027 and LVA348**Standard Deviation calculated from 2 independent tests (LVA027 andLVA348) ***Standard Deviation calculated from 4 independent tests****Corresponding to the following amounts of FIP U of reference lipaseLV2934: 500, 5000, 20000 and 75000 FIP Units determined by pancreaticFIP test, respectively

Additional lipase variants including LV1330, LV1855, LV1865, LV1874,LV1889, LVA043, LVA049, LVA012, LVA023, LVA099, LVA041, LVA061, LVA103,LV1857, LV1232, and LVA473 are studied in the same screening test.

All lipase variants tested were active in vivo and caused adose-dependent improvement in CFA. Lipase variants LVA129, LVA147,LVA238, LVA315, LVA317, LVA319, and LVA368 are all considerably improvedas compared to the reference lipase.

Example 11 Full In Vivo Digestibility Trial

The purified lipase variant LVA319 was tested in a full digestibilitystudy in a group of 6 female Göttingen minipigs (Ellegaard). Theefficacy has been compared to that of the lipase of SEQ ID NO: 1 testedin PEI minipigs fed the same diet. Pancreatic Exocrine Insufficiency(PEI) was induced in the minipigs by ligation of the pancreatic duct,and they were also fitted with an ileo-caecal re-entrant cannula, allunder isofluorane anaesthesia and at a weight of about 25 kg, asdescribed in Tabeling et al. (Tabeling et al., 1999, “Studies onnutrient digestibilities (pre-caecal and total) in pancreaticduct-ligated pigs and the effects of enzyme substitution”, J. Anim.Physiol. A. Anim. Nutr. 82: 251-263) and in Gregory et al. (Gregory etal., 1999, “Growth and digestion in pancreatic duct ligated pigs, Effectof enzyme supplementation” in “Biology of the Pancreas in GrowingAnimals” (Pierzynowski & Zabielski eds), Elsevier Science BV, Amsterdam,pp. 381-393). A period of at least 4 weeks was allowed for recovery fromsurgery, before studies were commenced. Prior to study begin, the PEIstatus of each pig was confirmed via the stool chymotrypsin test(commercially available from Immundiagnostik AG, Wiesenstrasse 4,D-64625 Bensheim, Germany, with catalogue No. K 6990).

Assay

During the studies, the pigs were housed in pens on a 12:12 h light-darkcycle and allowed free access to water and fed two meals per day.

Test Meal

During the study, the pigs were fed twice daily (08.00, 20.00 h) with300 g of a high-fat “human-like” diet containing: 200 g double-milleddiet (from Altromin), plus 25 g olive oil, 75 g cream and 0.625 g Cr₂O₃mixed with 1 litre water (see Table 13). The test meal contained 31%fat, 15% protein, 36% starch as well as vitamins, minerals and traceelements as per the nutritional requirements for pigs.

TABLE 13 Composition of experimental “human-like” diet Dietarycomponents Content (g/kg wet weight) Poultry meal 73 Pea meal 73 Casein(precipitated under acid conditions) 73 Wheat flour 290 Potato starch290 Lard 125 Vitamins, minerals, trace elements 76 To 200 g of the abovemixture were added: Cream (32% fat) 75 g Olive oil 25 g Chromic oxide0.625 g Water 1000 ml

The cream, and olive oil followed by tap water and finally the differentamounts/different enzyme supplements were mixed into the pre-weighed dryration (including the chromic oxide marker) shortly before the pigs werefed.

Performance

To assess lipase efficacy, the pigs were fed two 300 g test meals/dayinto which differing amounts of one or other of the two lipases weremixed immediately before feeding. The amount of each lipase administeredis shown in brackets in Table 15, viz. the activities in microbial FIP Ulipase/meal (lipase FIP units, see Example 1). Each enzyme dosage wasfed for 14 days: the pigs were fed the high-fat diet plus each newenzyme dosage for 9 days after which all faeces were collected over thenext 5 days, weighed and stored at −20° C. until analysis.

Analysis

The frozen faeces from each pig were freeze dried, weighed again andmilled. Aliquots of each of the 5 day milled samples (according to thedaily faecal production) were then pooled and mixed together; i.e.,giving one pooled sample for each pig for each dose of enzymes. Fromeach pooled sample the content of dry matter and crude fat weredetermined (Naumann & Bassler 1993; Die chemische Untersuchung vonFuttermitteln, 3. edition, VDLUFA-Verlag, Darmstadt (VDLUFA=VerbandDeutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten).Dry matter was estimated by weight after freeze-drying followed by 8 hincubation at 103° C.; crude fat content of the dried sample wasdetermined by acid hydrolysis and petrol ether extraction using a filterbag technique in an ANKOM^(XT15) extractor; Cr₂O₃ was oxidized tochromate and chromium content calculated as described by Petry and Rappin Zeitung für Tierphysiologie, 1970, vol. 27, p. 181-189. (Petry & Rapp1970; Z. Tierphysiol. 27; 181-189) via extinction at 365 nm(spectrophotometer).

Digestibility values (coefficient of fat absorption; CFA) were estimatedby the marker method according to the formula:

${C\; F\; A\mspace{14mu}(\%)} = {100 - \frac{\left\lbrack {\%\mspace{14mu}{Cr}_{2}O_{3}\mspace{14mu}{in}\mspace{14mu}{{feed} \cdot \%}\mspace{14mu}{fat}\mspace{14mu}{in}\mspace{14mu}{{faeces} \cdot 100}} \right\rbrack}{\left\lbrack {\%\mspace{14mu}{Cr}_{2}O_{3}\mspace{14mu}{in}\mspace{14mu}{{faeces} \cdot \%}\mspace{14mu}{fat}\mspace{14mu}{in}\mspace{14mu}{feed}} \right\rbrack}}$Results and Conclusion

From the results in Table 14 it is apparent that lipase variant LVA319performs much better than the reference lipase of SEQ ID NO: 1.

The lipase of the invention caused a very strong and dose-dependentimprovement in fat digestibility, already showing a highly efficientimprovement at the lowest dose tested.

TABLE 14 Influence of enzyme supplementation on CFA (Coefficient of FatAbsorption) Enzyme Supplement 0 Low Medium High No supplement 21.7 ± 4.5Reference lipase 46.3 +/− 4.9 59.2 +/− 7.0 75.6 +/− 4.7 (SEQ ID NO: 1)(40101 (155743 (1168069 FIP U) FIP U) FIP U) Lipase variant 59.2 +/− 4.472.0 +/− 4.3 81.4. +/− 1.3 LVA319  (9538  (38150  (114450 FIP U) FIP U)FIP U)

Example 12 Pharmaceutical Compositions

Pellets

A liquid lipase concentrate of purified lipase variant LVA129 (tested invivo in Example 10) is prepared. The liquid concentrate is dried byconventional means, and the lipase protein content of the dried powderis measured and should preferably lie above 50%. Then, 500 g driedlipase powder is dry pre-mixed together with 200 g microcrystallinecellulose and 300 g polyethylene glycol 4000 (Macrogol™ 4000) in acommercially available mixer. A sufficient amount of a commonly usedwetting agent is added and the resulting wet mass is thoroughly mixed atroom temperature. The homogenized mass is then extruded in acommercially available extruder fitted with a piercing die having a holediameter of 0.8 mm to form cylindrical pellets. The extrudate producedis rounded to spherical pellets with a commercially availablespheronizer by adding the necessary amount of a commonly used wettingagent. The pellets are dried at a product temperature of approximately40° C. in a commercially available vacuum dryer. The dried pellets arethen separated by using a mechanical sieving machine with 0.7 and 1.4 mmscreens. The sieve fractions of ≧0.7 mm and ≦1.4 mm are collected andfilled in portions of 200 mg pellets each in capsules of size 2.

The resulting pellets are tested for lipolytic activity by applying theLipase pH-stat assay described in Example 1.

The resulting pellets are tested for disintegration according to Pharm.Eur. 2.9.1. (Section “Disintegration of tablets and capsules”) (testsolution: 0.1 M malonic acid, pH 6.0-500 mL, 37° C.).

1. An isolated variant lipase which (a) has at least 90% sequenceidentity to the sequence of amino acids 1-269 of SEQ ID NO: 2; and (b)has lipase activity; and which, (c) as compared to the sequence of aminoacids 1-269 of SEQ ID NO: 2, comprises the four-fold substitutionN33Q+T231R+N233R+D254S and is selected from the following:N33Q+T231R+N233R+D254S; N33Q+D96N+T231R+N233R+D254S;N33Q+E210V+T231R+N233R+D254S; N11R+N33Q+E210V+T231R+N233R+D254S;N33Q+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+T231R+N233R+D254S;D27N+N33Q+G91T+G163K+T231R+N233R+D254S; N33Q+D111A+T231R+N233R+D254S;D27R+N33Q+G91A+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;N33Q+G91T+N94R+T231R+N233R+D254S; N33Q+W117L+T231R+N233R+D254S;N33Q+G91T+N94R+T231R+N233R+D254S; N33Q+L227F+T231R+N233R+D254S;D27R+N33Q+G91T+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;D27R+N33Q+G91T+D96E+D111A+T231R+N233R+D254S+P256T;D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;D27R+N33Q+G91N+N94R+D111A+S216P+L227G+T231R+N233R+D254S+P256T;D27R+N33Q+G91N+N94R+D111S+A155V+S216P+L227G+T231R+N233R+D254S+P256T;D27R+N33Q+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;N33Q+D111A+T231R+N233R+D254S; N33Q+D111A+W117L+T231R+N233R+D254S;N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;N33Q+G38A+G91T+G163K+T231R+N233R+D254S;D27V+N33Q+G91A+N94R+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;D27R+N33Q+P136H+L227G+T231R+N233R+Q249R+D254S;N33Q+G91T+D96N+D111A+V176I+T231R+N233R+D254S;N33Q+G91T+N94S+D111A+V176I+T231R+N233R+D254S;D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;D27R+N33Q+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;D27R+N33Q+G38A+G91T+D96E+D111A+T231R+N233R+D254S+P256T;N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;N11R+N33Q+G91T+W117I+G163K+T231R+N233R+D254S;D27R+N33Q+G38A+G91T+D96E+D111A+G163A+T231R+N233R+D254S+P256T;D27R+N33Q+V176Q+L227G+T231R+N233R+Q249R+D254S;N33Q+G91A+N94K+D111A+G163K+L227F+T231R+N233R+Q249R+D254S;N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;D27R+N33Q+W117I+V176Q+L227G+T231R+N233R+Q249R+D254S;N11R+N33Q+G38A+G91T+G163K+T231R+N233R+D254S;D27R+N33Q+G91T+D96E+L97Q+D111A+T231R+N233R+D254S+P256T;N11R+N33Q+G91T+S115L+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+V176W+T231R+N233R+D254S;E1D+N33Q+G91T+N94R+D111A+W117L+T231R+N233R+D254S;N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+D254S+P256T;D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+D254S+P256T;N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D242E+D254S+P256T;D27R+N33Q+G38A+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+D254S+P256T;N33Q+G38A+G91T+G163A+T231R+N233R+D254S;N11R+N33Q+190L+G163L+T231R+N233R+D254S;N11R+N33Q+E56Q+G91T+G163K+V176Q+T231R+N233R+D254S;N11R+D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;N11R+N33Q+G38A+G91T+G112A+G163A+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+E210D+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+V176T+T231R+N233R+D254S;N11R+N33Q+G91T+G163P+T231R+N233R+D254S;N11R+N33Q+G91M+G163T+T231R+N233R+D254S;N11R+N33Q+G38A+G91T+G163K+V176D+T231R+N233R+D254S;N11R+N33Q+E56Q+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+190L+G163K+T231R+N233R+D254S;D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+S216P+E219D+L227G+T231R+N233R+D254S+P256T;N11R+N33Q+190L+G91T+N94S+D96E+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+V176I+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+V176Q+T231R+N233R+D254S;N11R+N33Q+G91T+G163A+V176T+T231R+N233R+D254S;N11R+N33Q+G91T+G163L+V176I+T231R+N233R+D254S;N11R+N33Q+G91T+G163L+V176T+T231R+N233R+D254S;N11R+N33Q+G91T+G163L+T231R+N233R+D254S;N11R+N33Q+G91T+G163P+T231R+N233R+D254S;N11R+N33Q+G91T+G163P+V176I+T231R+N233R+D254S;N11R+N33Q+G91T+G163L+T231R+N233R+D254S+P256N;D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163T+S216P+L227G+T231R+N233R+Q249R+D254S+P256T;D27R+N33Q+L227G+T231R+N233R+Q249R+D254S;N33Q+G38A+G91T+G163K+T231R+N233R+D254S;N8L+N11R+N33Q+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+G38A+G91T+G163P+V176G+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256F;N11R+N33Q+G38A+G91T+G156R+G163K+V176T+T231R+N233R+D254S;N11R+N33Q+G91T+G163N+T231R+N233R+D254S;N11R+N33Q+G91T+G163T+T231R+N233R+D254S;N11R+N33Q+G91T+G163W+T231R+N233R+D254S;N11R+N33Q+G91K+G163K+T231R+N233R+D254S;N11R+G23E+N33Q+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+V141E+G163K+T231R+N233R+D254S;N11R+N33Q+L52R+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+V141L+G163K+T231R+N233R+D254S;N11R+N33Q+T37K+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+A68V+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+G163A+V176I+T231R+N233R+D254S;N11R+N33Q+T37M+G91T+G163P+V176T+T231R+N233R+D254S;N11R+N33Q+G91T+G163L+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+T231R+N233R+D254S+P256I;N11R+D27R+N33Q+E56Q+D57N+G91N+N94R+D111S+G163K+S216P+L227G+T231R+N233R+D254S+P256T;N11R+N33Q+G38A+G91T+G163P+V176G+T231R+N233R+D254S;N11R+N33Q+G38A+G91T+G163Q+V176G+T231R+N233R+D254S;N11R+N33Q+G38A+G91T+G163T+V176G+T231R+N233R+D254S;N11R+N33Q+G38A+G91T+N94R+G163P+V176G+T231R+N233R+D254S;E1*+N11R+N33Q+G38A+G91N+N94R+G163P+V176G+T231R+N233R+D254S;E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S;E1N+N33Q+G38A+G91T+G163P+V176F+L227F+T231R+N233R+D254S;E1N+N33Q+G38A+G91T+G163P+V176F+T231R+N233R+D254S+1255A+P256Q;E1N+N11R+N33Q+G38A+G91T+D111A+G163P+V176F+T231R+N233R+D254S;N11R+N33Q+G38A+G91T+D102G+S115L+G163K+T231R+N233R+D254S+P256T;N11R+N33Q+G38A+G91T+S115L+G163K+T231R+N233R+D254S+P256T;E1N+N11R+N33Q+G91T+G163A+T231R+N233R+G246A+D254S;N11R+D27R+N33Q+D57G+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T;N11C+N33Q+G91T+G163K+T231R+N233R+D254S;N11L+N33Q+G91T+G163K+T231R+N233R+D254S;N11H+N33Q+G91T+G163K+T231R+N233R+D254S;N11D+N33Q+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+D96W+G163K+T231R+N233R+D254S;D27R+N33Q+G91T+D96E+L97Q+D111A+G163K+T231R+N233R+D254S+P256T;N11P+N33Q+G91T+G163K+T231R+N233R+D254S;N11R+N33Q+E56Q+G163K+T231R+N233R+D254S;N11R+N33Q+G91T+G163A+T231R+N233R+D254S;N11R+N33Q+G91T+G163P+T231R+N233R+D254S;N11R+N33Q+G91T+G163K+L227G+P229R+T231R+N233R+D254S;N33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S;N33Q+G91T+K98I+G163K+T231R+N233R+D254S+P256L; andN33Q+G91T+T114I+G163K+T231R+N233R+D254S+P256L.
 2. The lipase of claim 1in combination with a protease or an amylase.
 3. The lipase of claim 1,in combination with a protease and an amylase.
 4. The lipase incombination with a protease or an amylase according to claim 2, wherein(i) the protease has at least 90% identity to a protease selected fromthe group consisting of a) a protease having the sequence of amino acids1-274 of SEQ ID NO: 3, b) a protease having the sequence of amino acids1-188 of SEQ ID NO: 4, and c) a protease having the sequence of aminoacids 1-188 of SEQ ID NO: 5; (ii) the amylase has at least 90% identityto an amylase selected from the group consisting of a) an amylase havingthe sequence of amino acids 1-481 of SEQ ID NO: 6, b) an amylase havingthe sequence of amino acids 1-481 of SEQ ID NO: 7, and c) an amylasehaving the sequence of amino acids 1-483 of SEQ ID NO:
 8. 5. Apharmaceutical composition comprising a lipase or a mixture of lipasesas defined in claim 1, together with at least one pharmaceuticallyacceptable auxiliary material.
 6. The composition of claim 5, furthercomprising a protease or an amylase.
 7. The composition of claim 5,further comprising a protease and an amylase.
 8. The composition ofclaim 5, wherein (i) the protease has at least 90% identity to aprotease selected from the group consisting of a) a protease having thesequence of amino acids 1-274 of SEQ ID NO: 3, b) a protease having thesequence of amino acids 1-188 of SEQ ID NO: 4, and c) a protease havingthe sequence of amino acids 1-188 of SEQ ID NO: 5; (ii) the amylase hasat least 90% identity to an amylase selected from the group consistingof a) an amylase having the sequence of amino acids 1-481 of SEQ ID NO:6, b) an amylase having the sequence of amino acids 1-481 of SEQ ID NO:7, and c) an amylase having the sequence of amino acids 1-483 of SEQ IDNO:
 8. 9. The lipase of claim 1, which is N33Q+T231R+N233R+D254S. 10.The lipase of claim 1, which is N11R+N33Q+G91T+G163K+T231R+N233R+D254S.11. The lipase of claim 1, which isN33Q+G91T+K98I+T114I+G163K+T231R+N233R+D254S.
 12. The lipase of claim 1,which is D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T.13. The lipase of claim 1, which isN11R+D27R+N33Q+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T.
 14. Amethod of treating a digestive disorder in an animal, comprisingadministering to the animal a therapeutically effective amount of thelipase of claim 1 and a pharmaceutically acceptable auxiliary material.15. The method of claim 14, wherein the digestive disorder ismaldigestion or dyspepsia.
 16. The method of claim 14, furthercomprising administering a protease or an amylase.
 17. A method oftreating pancreatic exocrine insufficiency, pancreatitis, cysticfibrosis, diabetes type I, and/or diabetes type II in an animal,comprising administering to the animal a therapeutically effectiveamount of the lipase of claim 1 and a pharmaceutically acceptableauxiliary material.
 18. The method of claim 17, wherein the pancreatitisis acute pancreatitis.
 19. The method of claim 17, wherein thepancreatitis is chronic pancreatitis.
 20. The method of claim 17,further comprising administering a protease or an amylase.
 21. Themethod of claim 14, further comprising administering a protease and anamylase.
 22. The method of claim 17, further comprising administering aprotease and an amylase.
 23. The lipase of claim 1, which has at least95% identity to the sequence of amino acids 1-269 of SEQ ID NO:
 2. 24.The lipase of claim 1, which has at least 97% identity to the sequenceof amino acids 1-269 of SEQ ID NO:
 2. 25. The lipase of claim 1, whichhas at least 99% identity to the sequence of amino acids 1-269 of SEQ IDNO: 2.